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1. Cleaving DNA with DNA: Cooperative Tuning of Structure and Reactivity Driven by Copper Ions

Author

Sarath Chandra Dantu, Mahdi Khalil, Marc Bria, Christine Saint‐Pierre, Maylis Orio, Didier Gasparutto, and Giuseppe Sicoli

Subjects
DNAzymes, hyperfine spectroscopy, metal soup, multiple binding, radical path, Science
Abstract

Abstract A copper‐dependent self‐cleaving DNA (DNAzyme or deoyxyribozyme) previously isolated by in vitro selection has been analyzed by a combination of Molecular Dynamics (MD) simulations and advanced Electron Paramagnetic Resonance (Electron Spin Resonance) EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction. The modeled 46‐nucleotide deoxyribozyme in MD simulations forms duplex and triplex sub‐structures that flank a highly conserved catalytic core. The DNA self‐cleaving construct can also form a bimolecular complex that has a distinct substrate and enzyme domains. The highly dynamic structure combined with an oxidative site‐specific cleavage of the substrate are two key‐aspects to elucidate. By combining EPR/ESR spectroscopy with selectively isotopically labeled nucleotides it has been possible to overcome the major drawback related to the “metal‐soup” scenario, also known as “super‐stoichiometric” ratios of cofactors versus substrate, conventionally required for the DNA cleavage reaction within those nucleic acids‐based enzymes. The focus on the endogenous paramagnetic center (Cu2+) here described paves the way for analysis on mixtures where several different cofactors are involved. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic perspective towards the applicability of EPR/ESR spectroscopic studies.

Published
2024
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2. In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans

Author

Anna Seck, Salvatore De Bonis, Christine Saint-Pierre, Didier Gasparutto, Jean-Luc Ravanat, and Joanna Timmins

Subjects
Biology (General), QH301-705.5
Abstract

Reconstitution of D radiodurans nucleotide excision repair provides insights into the kinetics of repair on different DNA substrates and determines the order and precise sites of incisions on the 5’ and 3’ sides of the lesion.

Published
2022
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3. The Arabidopsis thaliana Poly(ADP-Ribose) Polymerases 1 and 2 Modify DNA by ADP-Ribosylating Terminal Phosphate Residues

Author

Sabira Taipakova, Aigerim Kuanbay, Christine Saint-Pierre, Didier Gasparutto, Yeldar Baiken, Regina Groisman, Alexander A. Ishchenko, Murat Saparbaev, and Amangeldy K. Bissenbaev

Subjects
plant DNA repair, Arabidopsis thaliana, DNA strand break, nicotinamide adenine dinucleotide (NAD+), poly(ADP-ribose) polymerase (PARP), ADP-ribosylation, Biology (General), QH301-705.5
Abstract

Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyze the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are viewed as DNA damage sensors that, upon binding to strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. The flowering plant Arabidopsis thaliana contains three genes encoding homologs of mammalian PARPs: atPARP1, atPARP2, and atPARP3. Both atPARP1 and atPARP2 contain poly(ADP-ribosyl)ating activity; however, it is unknown whether they could covalently modify DNA by ADP-ribosylating the strand break termini. Here, we report that similar to their mammalian counterparts, the plant atPARP1 and atPARP2 proteins ADP-ribosylate 5′-terminal phosphate residues in duplex DNA oligonucleotides and plasmid containing at least two closely spaced DNA strand breaks. AtPARP1 preferentially catalyzes covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 5′-phosphate, whereas atPARP2 preferentially ADP-ribosylates the nicked and gapped DNA duplexes containing the terminal 5′-phosphate. Similar to their mammalian counterparts, the plant PARP-catalyzed DNA ADP-ribosylation is particularly sensitive to the distance that separates two strand breaks in the same DNA molecule, 1.5 and 1 or 2 turns of helix for atPARP1 and atPARP2, respectively. PAR glycohydrolase (PARG) restored native DNA structure by hydrolyzing the PAR–DNA adducts generated by atPARPs. Biochemical and mass spectrometry analyses of the PAR–DNA adducts showed that atPARPs utilize phosphorylated DNA termini as an alternative to protein acceptor residues to catalyze PAR chain synthesis via phosphodiester bond formation between C1′ of ADP-ribose and a phosphate residue of the terminal nucleotide in DNA fragment. Taken together, these data establish the presence of a new type of DNA-modifying activity in Arabidopsis PARPs, suggesting a possible role of DNA ADP-ribosylation in DNA damage signaling and repair of terrestrial plants.

Published
2020
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4. Dissecting Highly Mutagenic Processing of Complex Clustered DNA Damage in Yeast Saccharomyces cerevisiae

Author

Stanislav G. Kozmin, Gregory Eot-Houllier, Anne Reynaud-Angelin, Didier Gasparutto, and Evelyne Sage

Subjects
clustered DNA damage, multiply damaged sites, non-DSB clustered DNA damage, oxidized base lesions, base excision repair, mutagenesis, Cytology, QH573-671
Abstract

Clusters of DNA damage, also called multiply damaged sites (MDS), are a signature of ionizing radiation exposure. They are defined as two or more lesions within one or two helix turns, which are created by the passage of a single radiation track. It has been shown that the clustering of DNA damage compromises their repair. Unresolved repair may lead to the formation of double-strand breaks (DSB) or the induction of mutation. We engineered three complex MDS, comprised of oxidatively damaged bases and a one-nucleotide (1 nt) gap (or not), in order to investigate the processing and the outcome of these MDS in yeast Saccharomyces cerevisiae. Such MDS could be caused by high linear energy transfer (LET) radiation. Using a whole-cell extract, deficient (or not) in base excision repair (BER), and a plasmid-based assay, we investigated in vitro excision/incision at the damaged bases and the mutations generated at MDS in wild-type, BER, and translesion synthesis-deficient cells. The processing of the studied MDS did not give rise to DSB (previously published). Our major finding is the extremely high mutation frequency that occurs at the MDS. The proposed processing of MDS is rather complex, and it largely depends on the nature and the distribution of the damaged bases relative to the 1 nt gap. Our results emphasize the deleterious consequences of MDS in eukaryotic cells.

Published
2021
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5. Aberrant repair initiated by the adenine-DNA glycosylase does not play a role in UV-induced mutagenesis in Escherichia coli

Author

Caroline Zutterling, Aibek Mursalimov, Ibtissam Talhaoui, Zhanat Koshenov, Zhiger Akishev, Amangeldy K. Bissenbaev, Gerard Mazon, Nicolas E. Geacintov, Didier Gasparutto, Regina Groisman, Dmitry O. Zharkov, Bakhyt T. Matkarimov, and Murat Saparbaev

Subjects
Adenine-DNA glycosylase, Nucleotide excision repair, UV-induced mutagenesis, Cyclobutane pyrimidine dimer, Base excision repair, Aberrant DNA repair, Medicine, Biology (General), QH301-705.5
Abstract

Background DNA repair is essential to counteract damage to DNA induced by endo- and exogenous factors, to maintain genome stability. However, challenges to the faithful discrimination between damaged and non-damaged DNA strands do exist, such as mismatched pairs between two regular bases resulting from spontaneous deamination of 5-methylcytosine or DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved the mismatch-specific DNA glycosylases that can recognize and remove regular DNA bases in the mismatched DNA duplexes. The Escherichia coli adenine-DNA glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2′-deoxyguanosine-5′-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T→C•G transversions. Furthermore, it has been demonstrated that human MUTYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage. Methods Here, we investigated the role of MutY in UV-induced mutagenesis in E. coli. MutY was probed on DNA duplexes containing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproduct (6–4PP). UV irradiation of E. coli induces Save Our Souls (SOS) response characterized by increased production of DNA repair enzymes and mutagenesis. To study the role of MutY in vivo, the mutation frequencies to rifampicin-resistant (RifR) after UV irradiation of wild type and mutant E. coli strains were measured. Results We demonstrated that MutY does not excise Adenine when it is paired with CPD and 6–4PP adducts in duplex DNA. At the same time, MutY excises Adenine in A•G and A•8oxoG mispairs. Interestingly, E. coli mutY strains, which have elevated spontaneous mutation rate, exhibited low mutational induction after UV exposure as compared to MutY-proficient strains. However, sequence analysis of RifR mutants revealed that the frequencies of C→T transitions dramatically increased after UV irradiation in both MutY-proficient and -deficient E. coli strains. Discussion These findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damage.

Published
2018
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6. Melting Curve Analysis of Aptachains: Adenosine Detection with Internal Calibration

Author

Chenze Lu, Christine Saint-Pierre, Didier Gasparutto, Yoann Roupioz, Corinne Ravelet, Eric Peyrin, and Arnaud Buhot

Subjects
split-aptamers, small molecule detection, aptachain self-assembly, melting temperature, calibration/normalization, Biotechnology, TP248.13-248.65
Abstract

Small molecules are ubiquitous in nature and their detection is relevant in various domains. However, due to their size, sensitive and selective probes are difficult to select and the detection methods are generally indirect. In this study, we introduced the use of melting curve analysis of aptachains based on split-aptamers for the detection of adenosine. Aptamers, short oligonucleotides, are known to be particularly efficient probes compared to antibodies thanks to their advantageous probe/target size ratio. Aptachains are formed from dimers with dangling ends followed by the split-aptamer binding triggered by the presence of the target. The high melting temperature of the dimers served as a calibration for the detection/quantification of the target based on the height and/or temperature shift of the aptachain melting peak.

Published
2021
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7. Solution-phase vs surface-phase aptamer-protein affinity from a label-free kinetic biosensor.

Author

Camille Daniel, Yoann Roupioz, Didier Gasparutto, Thierry Livache, and Arnaud Buhot

Subjects
Medicine, Science
Abstract

Aptamers are selected DNA ligands that target biomolecules such as proteins. In recent years, they are showing an increasing interest as potential therapeutic agents or recognition elements in biosensor applications. In both cases, the need for characterizing the mating between the target and the aptamer either in solution or immobilized on a surface, is pressing. In this context, we have developed a kinetic biosensor made of micro-arrayed anti-thrombin aptamers to assess the kinetic parameters of this interaction. The binding of label-free thrombin on the biosensor was monitored in real-time by Surface Plasmon Resonance imaging. Remarkable performances were obtained for the quantification of thrombin without amplification (sub-nanomolar limit of detection and linear range of quantification to two orders of magnitude). The independent determinations of both the solution- and surface-phase affinities, respectively KD(Sol) and KD(Surf), revealed distinct values illustrating the importance of probes, targets or surface interactions in biosensors. Interestingly, KD(Surf) values depend on the aptamer grafting density and linearly extrapolate towards KD(Sol) for highly diluted probes. This suggests a lesser impact of the surface compared to the probe or target cooperativity interactions since the latter decrease with a reduced grafting density.

Published
2013
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9. Highly mutagenic exocyclic DNA adducts are substrates for the human nucleotide incision repair pathway.

Author

Paulina Prorok, Christine Saint-Pierre, Didier Gasparutto, Olga S Fedorova, Alexander A Ishchenko, Hervé Leh, Malcolm Buckle, Barbara Tudek, and Murat Saparbaev

Subjects
Medicine, Science
Abstract

BACKGROUND: Oxygen free radicals induce lipid peroxidation (LPO) that damages and breaks polyunsaturated fatty acids in cell membranes. LPO-derived aldehydes and hydroxyalkenals react with DNA leading to the formation of etheno(ε)-bases including 1,N(6)-ethenoadenine (εA) and 3,N(4)-ethenocytosine (εC). The εA and εC residues are highly mutagenic in mammalian cells and eliminated in the base excision repair (BER) pathway and/or by AlkB family proteins in the direct damage reversal process. BER initiated by DNA glycosylases is thought to be the major pathway for the removal of non-bulky endogenous base damage. Alternatively, in the nucleotide incision repair (NIR) pathway, the apurinic/apyrimidinic (AP) endonucleases can directly incise DNA duplex 5' to a damaged base in a DNA glycosylase-independent manner. METHODOLOGY/PRINCIPAL FINDINGS: Here we have characterized the substrate specificity of human major AP endonuclease 1, APE1, towards εA, εC, thymine glycol (Tg) and 7,8-dihydro-8-oxoguanine (8oxoG) residues when present in duplex DNA. APE1 cleaves oligonucleotide duplexes containing εA, εC and Tg, but not those containing 8oxoG. Activity depends strongly on sequence context. The apparent kinetic parameters of the reactions suggest that APE1 has a high affinity for DNA containing ε-bases but cleaves DNA duplexes at an extremely slow rate. Consistent with this observation, oligonucleotide duplexes containing an ε-base strongly inhibit AP site nicking activity of APE1 with IC(50) values in the range of 5-10 nM. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of εA•T and εC•G duplexes generates, as expected, DNA fragments containing 5'-terminal ε-base residue. CONCLUSIONS/SIGNIFICANCE: The fact that ε-bases and Tg in duplex DNA are recognized and cleaved by APE1 in vitro, suggests that NIR may act as a backup pathway to BER to remove a large variety of genotoxic base lesions in human cells.

Published
2012
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10. Unexpected role for Helicobacter pylori DNA polymerase I as a source of genetic variability.

Author

María-Victoria García-Ortíz, Stéphanie Marsin, Mercedes E Arana, Didier Gasparutto, Raphaël Guérois, Thomas A Kunkel, and J Pablo Radicella

Subjects
Genetics, QH426-470
Abstract

Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in DNA repair. We report that H. pylori DNA Pol I 5'- 3' exonuclease domain is essential for viability, probably through its involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA, it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity.

Published
2011
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11. New insights in the removal of the hydantoins, oxidation product of pyrimidines, via the base excision and nucleotide incision repair pathways.

Author

Modesto Redrejo-Rodríguez, Christine Saint-Pierre, Sophie Couve, Abdelghani Mazouzi, Alexander A Ishchenko, Didier Gasparutto, and Murat Saparbaev

Subjects
Medicine, Science
Abstract

BACKGROUND: Oxidative damage to DNA, if not repaired, can be both miscoding and blocking. These genetic alterations can lead to mutations and/or cell death, which in turn cause cancer and aging. Oxidized DNA bases are substrates for two overlapping repair pathways: base excision (BER) and nucleotide incision repair (NIR). Hydantoin derivatives such as 5-hydroxyhydantoin (5OH-Hyd) and 5-methyl-5-hydroxyhydantoin (5OH-5Me-Hyd), major products of cytosine and thymine oxidative degradation pathways, respectively, have been detected in cancer cells and ancient DNA. Hydantoins are blocking lesions for DNA polymerases and excised by bacterial and yeast DNA glycosylases in the BER pathway. However little is known about repair of pyrimidine-derived hydantoins in human cells. METHODOLOGY/PRINCIPAL FINDINGS: Here, using both denaturing PAGE and MALDI-TOF MS analyses we report that the bacterial, yeast and human AP endonucleases can incise duplex DNA 5' next to 5OH-Hyd and 5OH-5Me-Hyd thus initiating the NIR pathway. We have fully reconstituted the NIR pathway for these lesions in vitro using purified human proteins. Depletion of Nfo in E. coli and APE1 in HeLa cells abolishes the NIR activity in cell-free extracts. Importantly, a number of redundant DNA glycosylase activities can excise hydantoin residues, including human NTH1, NEIL1 and NEIL2 and the former protein being a major DNA glycosylase activity in HeLa cells extracts. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that both BER and NIR pathways can compete and/or back-up each other to remove hydantoin DNA lesions in vivo.

Published
2011
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12. Structural and functional insights into the activation of the dual incision activity of UvrC, a key player in bacterial NER

Author

Anna Seck, Salvatore De Bonis, Meike Stelter, Mats Ökvist, Müge Senarisoy, Mohammad Rida Hayek, Aline Le Roy, Lydie Martin, Christine Saint-Pierre, Célia M Silveira, Didier Gasparutto, Smilja Todorovic, Jean-Luc Ravanat, Joanna Timmins, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), European Synchrotron Radiation Facility (ESRF), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Instituto de Tecnologia Química e Biológica António Xavier (ITQB), and Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)

Subjects
MESH: DNA Damage, MESH: DNA Repair, MESH: Escherichia coli, [SDV]Life Sciences [q-bio], Genetics, MESH: DNA Helicases, MESH: Endodeoxyribonucleases, MESH: Bacterial Proteins, MESH: DNA, Bacterial, MESH: Deinococcus
Abstract

Bacterial nucleotide excision repair (NER), mediated by the UvrA, UvrB and UvrC proteins is a multistep, ATP-dependent process, that is responsible for the removal of a very wide range of chemically and structurally diverse DNA lesions. DNA damage removal is performed by UvrC, an enzyme possessing a dual endonuclease activity, capable of incising the DNA on either side of the damaged site to release a short single-stranded DNA fragment containing the lesion. Using biochemical and biophysical approaches, we have probed the oligomeric state, UvrB- and DNA-binding abilities and incision activities of wild-type and mutant constructs of UvrC from the radiation resistant bacterium, Deinococcus radiodurans. Moreover, by combining the power of new structure prediction algorithms and experimental crystallographic data, we have assembled the first model of a complete UvrC, revealing several unexpected structural motifs and in particular, a central inactive RNase H domain acting as a platform for the surrounding domains. In this configuration, UvrC is maintained in a ‘closed’ inactive state that needs to undergo a major rearrangement to adopt an ‘open’ active state capable of performing the dual incision reaction. Taken together, this study provides important insight into the mechanism of recruitment and activation of UvrC during NER.

Published
2023

13. Photoactivatable V-Shaped Bifunctional Quinone Methide Precursors as a New Class of Selective G-quadruplex Alkylating Agents

Author

Alberto Lena, Alessandra Benassi, Michele Stasi, Christine Saint‐Pierre, Mauro Freccero, Didier Gasparutto, Sophie Bombard, Filippo Doria, Daniela Verga, SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Université Paris-Saclay, Institut Curie [Paris], ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), and ANR-19-CE44-0001,NEMESES,Identification à l'échelle du génome des sites de fixation à la base exacte des ligands de l'ADN G4(2019)

Subjects
G-Quadruplexes, Alkylating Agents, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Organic Chemistry, [CHIM]Chemical Sciences, Humans, General Chemistry, Indolequinones, Ligands, Catalysis
Abstract

International audience; Combining the selectivity of G-quadruplex (G4) ligands with the spatial and temporal control of photochemistry is an emerging strategy to elucidate the biological relevance of these structures. In this work, we developed six novel V-shaped G4 ligands that can, upon irradiation, form stable covalent adducts with G4 structures via the reactive intermediate, quinone methide (QM). We thoroughly investigated the photochemical properties of the ligands and their ability to generate QMs. Subsequently, we analysed their specificity for various topologies of G4 and discovered a preferential binding towards the human telomeric sequence. Finally, we tested the ligand ability to act as photochemical alkylating agents, identifying the covalent adducts with G4 structures. This work introduces a novel molecular tool in the chemical biology toolkit for G4s.

Published
2022

14. Hydrogen Production at a NiO Photocathode Based on a Ruthenium Dye-Cobalt Diimine Dioxime Catalyst Assembly: Insights from Advanced Spectroscopy and Post-operando Characterization

Author

Didier Gasparutto, Benjamin Dietzek, Alexander Schwab, Murielle Chavarot-Kerlidou, Stephan Kupfer, Didier Léonard, Christine Saint-Pierre, Sebastian Bold, Nicolas Queyriaux, Jakob Bruhnke, Carolin Müller, Emmanouil Giannoudis, Vincent Artero, Dmitry Aldakov, Corinne Gablin, Solar fuels, hydrogen and catalysis (SolHyCat), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller-Universität = Friedrich Schiller University Jena [Jena, Germany], Department Functional Interfaces, Leibniz Institute of Photonic Technology Jena (IPHT), Center for Energy and Environmental Chemistry (CEEC Jena), Institut des Sciences Analytiques (ISA), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Synthèse, Structure et Propriétés de Matériaux Fonctionnels (STEP ), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Leibniz Institute of Photonic Technology Jena, Department Functional Interfaces, Albert-Einstein-Straße 9, 07745 Jena, Germany, Institute of Physical Chemistry and Center for Energy and Environmental Chemistry Jena, (CEEC- Jena), Surfaces, Centre National de la Recherche Scientifique (CNRS)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Institute for Physical Chemistry and Center for Energy and Environmental Chemistry, This work was supported by the European Commission's Seventh Framework Program (FP7/2007-2013) under grant agreement no. 306398 (FP7-IDEAS-ERC, Project PhotocatH2ode), the Deutsche Forschungsgemeinschaft (German Science Foundation, PHOTOACC project, grant no. KU 3933/2-1), and the French National Research Agency in the framework of the 'Investissements d'avenir' program (ANR15-IDEX-02, Labex ARCANE and CBH-EURGS, ANR-17EURE-0003) and the Franco-German University., ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), European Project: 306398,EC:FP7:ERC,ERC-2012-StG_20111012,PHOTOCATH2ODE(2012), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
dye-sensitized, Materials science, Hydrogen, solar fuels, Photoelectrochemistry, chemistry.chemical_element, 02 engineering and technology, [CHIM.INOR]Chemical Sciences/Inorganic chemistry, 010402 general chemistry, Photochemistry, 7. Clean energy, 01 natural sciences, Catalysis, Artificial photosynthesis, photoelectrochemistry, cobalt catalyst, General Materials Science, Hydrogen production, [CHIM.MATE]Chemical Sciences/Material chemistry, [CHIM.CATA]Chemical Sciences/Catalysis, 021001 nanoscience & nanotechnology, Solar fuel, 0104 chemical sciences, Ruthenium, chemistry, 13. Climate action, hydrogen, [PHYS.COND.CM-MS]Physics [physics]/Condensed Matter [cond-mat]/Materials Science [cond-mat.mtrl-sci], Water splitting, 0210 nano-technology, [CHIM.OTHE]Chemical Sciences/Other
Abstract

International audience; The production of hydrogen by efficient, low-cost, and integrated photoelectrochemical water splitting processes represents an important target for the ecological transition. This challenge can be addressed thanks to bioinspired chemistry and artificial photosynthesis approaches by designing dye-sensitized photocathodes for hydrogen production, incorporating bioinspired first-row transition metal-based catalysts. The present work r describes the preparation and photoelectrochemical characterization of a NiO photocathode sensitized with a phosphonate-derivatized ruthenium tris-diimine photosensitizer covalently linked to a cobalt diimine dioxime hydrogen-evolving catalyst. Under simulated AM 1.5G irradiation, hydrogen is produced with photocurrent densities reaching 84 +/- 7 mu A.cm(-2), which is among the highest values reported so far for dye-sensitized photocathodes with surface-immobilized catalysts. Thanks to the unique combination of advanced spectroscopy and surface characterization techniques, the fast desorption of the dyad from the NiO electrode and the low yield of electron transfer to the catalyst, resulting in the Co demetallation from the diimine dioxime framework, were identified as the main barriers limiting the performances and the stability of the system. This work therefore paves the way for a more rational design of molecular photocathodes for solar fuel production and represents a further step toward the development of sustainable processes for the production of hydrogen from sunlight and water.

Published
2021

15. In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans

Author

Anna Seck, Salvatore De Bonis, Christine Saint-Pierre, Didier Gasparutto, Jean-Luc Ravanat, Joanna Timmins, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Grenoble Instruct-ERIC center (ISBG, UMS 3518 CNRS-CEA-UGA-EMBL), Grenoble Partnership for Structural Biology (PSB), GRAL LABEX, ARCANE LABEX, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

Subjects
MESH: DNA Damage, MESH: DNA Repair, Endodeoxyribonucleases, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], DNA Repair, QH301-705.5, Escherichia coli Proteins, Medicine (miscellaneous), MESH: Escherichia coli Proteins, MESH: Deinococcus, General Biochemistry, Genetics and Molecular Biology, MESH: Endodeoxyribonucleases, Deinococcus, Biology (General), General Agricultural and Biological Sciences, DNA Damage
Abstract

Nucleotide excision repair (NER) is a universal and versatile DNA repair pathway, capable of removing a very wide range of lesions, including UV-induced pyrimidine dimers and bulky adducts. In bacteria, NER involves the sequential action of the UvrA, UvrB and UvrC proteins to release a short 12- or 13-nucleotide DNA fragment containing the damaged site. Although bacterial NER has been the focus of numerous studies over the past 40 years, a number of key questions remain unanswered regarding the mechanisms underlying DNA damage recognition by UvrA, the handoff to UvrB and the site-specific incision by UvrC. In the present study, we have successfully reconstituted in vitro a robust NER system using the UvrABC proteins from the radiation resistant bacterium, Deinococcus radiodurans. We have investigated the influence of various parameters, including temperature, salt, protein and ATP concentrations, protein purity and metal cations, on the dual incision by UvrABC, so as to find the optimal conditions for the efficient release of the short lesion-containing oligonucleotide. This newly developed assay relying on the use of an original, doubly-labelled DNA substrate has allowed us to probe the kinetics of repair on different DNA substrates and to determine the order and precise sites of incisions on the 5′ and 3′ sides of the lesion. This new assay thus constitutes a valuable tool to further decipher the NER pathway in bacteria.

Published
2021

16. The

Author

Sabira, Taipakova, Aigerim, Kuanbay, Christine, Saint-Pierre, Didier, Gasparutto, Yeldar, Baiken, Regina, Groisman, Alexander A, Ishchenko, Murat, Saparbaev, and Amangeldy K, Bissenbaev

Subjects
DNA strand break, Cell and Developmental Biology, Arabidopsis thaliana, plant DNA repair, nicotinamide adenine dinucleotide (NAD+), Original Research, poly(ADP-ribose) polymerase (PARP), ADP-ribosylation
Abstract

Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyze the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are viewed as DNA damage sensors that, upon binding to strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. The flowering plant Arabidopsis thaliana contains three genes encoding homologs of mammalian PARPs: atPARP1, atPARP2, and atPARP3. Both atPARP1 and atPARP2 contain poly(ADP-ribosyl)ating activity; however, it is unknown whether they could covalently modify DNA by ADP-ribosylating the strand break termini. Here, we report that similar to their mammalian counterparts, the plant atPARP1 and atPARP2 proteins ADP-ribosylate 5′-terminal phosphate residues in duplex DNA oligonucleotides and plasmid containing at least two closely spaced DNA strand breaks. AtPARP1 preferentially catalyzes covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 5′-phosphate, whereas atPARP2 preferentially ADP-ribosylates the nicked and gapped DNA duplexes containing the terminal 5′-phosphate. Similar to their mammalian counterparts, the plant PARP-catalyzed DNA ADP-ribosylation is particularly sensitive to the distance that separates two strand breaks in the same DNA molecule, 1.5 and 1 or 2 turns of helix for atPARP1 and atPARP2, respectively. PAR glycohydrolase (PARG) restored native DNA structure by hydrolyzing the PAR–DNA adducts generated by atPARPs. Biochemical and mass spectrometry analyses of the PAR–DNA adducts showed that atPARPs utilize phosphorylated DNA termini as an alternative to protein acceptor residues to catalyze PAR chain synthesis via phosphodiester bond formation between C1′ of ADP-ribose and a phosphate residue of the terminal nucleotide in DNA fragment. Taken together, these data establish the presence of a new type of DNA-modifying activity in Arabidopsis PARPs, suggesting a possible role of DNA ADP-ribosylation in DNA damage signaling and repair of terrestrial plants.

Published
2020

17. Aqueous Synthesis of DNA-Functionalized Near-Infrared AgInS

Author

Annette, Delices, Davina, Moodelly, Charlotte, Hurot, Yanxia, Hou, Wai Li, Ling, Christine, Saint-Pierre, Didier, Gasparutto, Gilles, Nogues, Peter, Reiss, and Kuntheak, Kheng

Subjects
Silver, Infrared Rays, Surface Properties, Zinc Compounds, Quantum Dots, Water, DNA, Particle Size, Sulfides, Indium, Sulfur
Abstract

Biocompatibility, biofunctionality, and chemical stability are essential criteria to be fulfilled by quantum dot (QD) emitters for bio-imaging and -sensing applications. In addition to these criteria, achieving efficient near-infrared (NIR) emission with nontoxic QDs remains very challenging. In this perspective, we developed water-soluble NIR-emitting AgInS

Published
2020

18. Solid-phase synthesis of branched oligonucleotides containing a biologically relevant dCyd341 interstrand crosslink DNA lesion

Author

Didier Gasparutto, Christine Saint-Pierre, Jean-Luc Ravanat, Marisa L Taverna Porro, Chimie Interface Biologie pour l’Environnement, la Santé et la Toxicologie (CIBEST ), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Département Interfaces pour l'énergie, la Santé et l'Environnement (DIESE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

Subjects
Exonuclease, Stereochemistry, Biochemistry, DNA DAMAGE, purl.org/becyt/ford/1 [https], 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Solid-phase synthesis, Ribose, purl.org/becyt/ford/1.4 [https], Physical and Theoretical Chemistry, Protecting group, BRANCHED OLIGONUCLEOTIDE, Solid-Phase Synthesis Techniques, 030304 developmental biology, INTERSTRAND CROSS-LINKS, 0303 health sciences, Phosphoramidite, biology, Oligonucleotide, Organic Chemistry, chemistry, Oligodeoxyribonucleotides, 030220 oncology & carcinogenesis, biology.protein, [CHIM.RADIO]Chemical Sciences/Radiochemistry, Cytosine, DNA, DNA Damage
Abstract

Branched oligonucleotides containing a biologically relevant DNA lesion, dCyd341, which involves an interstrand crosslink between a cytosine base on one strand and a ribose moiety on the opposite strand, were prepared in a single automated solid-phase synthesis. For this, we first prepared the phosphoramidite analogue of dCyd341 bearing an orthogonal levulinyl protecting group. Then, following the synthesis of the first DNA strand containing dCyd341, the levulinic group was removed and the synthesis was then continued from the free base hydroxyl group at the branching point, using traditional phosphoramidites. The synthesized oligonucleotides were fully characterized by MALDI-TOF/MS and were enzymatically digested, and the presence of the lesion was confirmed by HPLC-MS/MS and the sequence was finally controlled upon exonuclease digestion followed by MALDI-TOF/MS analysis. The developed strategy was successfully employed for the preparation of several short linear and branched oligonucleotides containing the aforementioned lesion. Fil: Taverna Porro, Marisa Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; Argentina Fil: Saint Pierre, Christine. Universite Grenoble Alpes; Francia Fil: Gasparutto, Didier. Universite Grenoble Alpes; Francia Fil: Ravanat, Jean Luc. Universite Grenoble Alpes; Francia

Published
2020

19. Aqueous Synthesis of DNA-Functionalized Near-Infrared AgInS 2 /ZnS Core/Shell Quantum Dots

Author

Wai Li Ling, Gilles Nogues, Annette Delices, Kuntheak Kheng, Peter Reiss, Charlotte Hurot, Yanxia Hou, Christine Saint-Pierre, Davina Moodelly, Didier Gasparutto, Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB ), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Département Interfaces pour l'énergie, la Santé et l'Environnement (DIESE), PHotonique, ELectronique et Ingénierie QuantiqueS (PHELIQS), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Synthèse, Structure et Propriétés de Matériaux Fonctionnels (STEP ), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Nanophysique et Semiconducteurs (NPSC), Institut Néel (NEEL), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), Université Grenoble Alpes (UGA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Nanophysique et Semiconducteurs (NEEL - NPSC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-16-CE09-0015,NEUTRINOS,Suivi des interactions biologiques par détection optique ultrasensible à base de nanoparticules(2016), and ANR-18-CE09-0039,FLUO,Synthèse en flux continu de quantum dots à base d'InP(2018)

Subjects
Photoluminescence, Materials science, Biocompatibility, AgInS2, Quantum yield, 02 engineering and technology, 010402 general chemistry, Photochemistry, 01 natural sciences, [CHIM]Chemical Sciences, General Materials Science, Surface plasmon resonance, hot-injection, ComputingMilieux_MISCELLANEOUS, Bioconjugation, Quantum dots, technology, industry, and agriculture, DNA functionalization, semiconductor nanocrystals, equipment and supplies, 021001 nanoscience & nanotechnology, 0104 chemical sciences, core-shell, seed-mediated growth, Quantum dot, Chemical stability, 0210 nano-technology, Luminescence, surface plasmon resonance
Abstract

Biocompatibility, biofunctionality, and chemical stability are essential criteria to be fulfilled by quantum dot (QD) emitters for bio-imaging and -sensing applications. In addition to these criteria, achieving efficient near-infrared (NIR) emission with nontoxic QDs remains very challenging. In this perspective, we developed water-soluble NIR-emitting AgInS2/ZnS core/shell (AIS/ZnS) QDs functionalized with DNA. The newly established aqueous route relying on a two-step hot-injection synthesis led to highly luminescent chalcopyrite-type AIS/ZnS core/shell QDs with an unprecedented photoluminescence quantum yield (PLQY) of 55% at 700 nm and a long photoluminescence (PL) decay time of 900 ns. Fast and slow hot injection of the precursors were compared for the AIS core QD synthesis, yielding a completely different behavior in terms of size, size distribution, stoichiometry, and crystal structure. The PL peak positions of both types of core QDs were 710 (fast) and 760 nm (slow injection) with PLQYs of 36 and 8%, respectively. The slow and successive incorporation of the Zn and S precursors during the subsequent shell growth step on the stronger emitting cores promoted the formation of a three-monolayer thick ZnS shell, evidenced by the increase of the average QD size from 3.0 to 4.8 nm. Bioconjugation of the AIS/ZnS QDs with hexylthiol-modified DNA was achieved during the ZnS shell growth, resulting in a grafting level of 5-6 DNA single strands per QD. The successful chemical conjugation of DNA was attested by UV-vis spectroscopy and agarose gel electrophoresis. Importantly, surface plasmon resonance imaging experiments using complementary DNA strands further corroborated the successful coupling and the stability of the AIS/ZnS-DNA QD conjugates as well as the preservation of the biological activity of the anchored DNA. The strong NIR emission and biocompatibility of these AIS/ZnS-DNA QDs provide a high potential for their use in biomedical applications.

Published
2020

20. Melting Curve Analysis of Aptachains: Adenosine Detection with Internal Calibration

Author

Didier Gasparutto, Christine Saint-Pierre, Corinne Ravelet, Arnaud Buhot, Yoann Roupioz, Chenze Lu, Eric Peyrin, SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Grenoble Alpes (UGA), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

Subjects
aptachain self-assembly, Adenosine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Materials science, Polymers, lcsh:Biotechnology, Melting temperature, Aptamer, calibration/normalization, Clinical Biochemistry, Analytical chemistry, Biosensing Techniques, 010402 general chemistry, split-aptamers, melting temperature, 01 natural sciences, Article, small molecule detection, Melting curve analysis, lcsh:TP248.13-248.65, Calibration, medicine, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Oligonucleotide, 010401 analytical chemistry, General Medicine, Aptamers, Nucleotide, Small molecule, 0104 chemical sciences, Size ratio, medicine.drug
Abstract

International audience; Small molecules are ubiquitous in nature and their detection is relevant in various domains. However, due to their size, sensitive and selective probes are difficult to select and the detection methods are generally indirect. In this study, we introduced the use of melting curve analysis of aptachains based on split-aptamers for the detection of adenosine. Aptamers, short oligonucleotides, are known to be particularly efficient probes compared to antibodies thanks to their advantageous probe/target size ratio. Aptachains are formed from dimers with dangling ends followed by the split-aptamer binding triggered by the presence of the target. The high melting temperature of the dimers served as a calibration for the detection/quantification of the target based on the height and/or temperature shift of the aptachain melting peak.

Published
2021

21. DNA Origami Mask for Sub-Ten-Nanometer Lithography

Author

Cheikh Tidiane Diagne, Xavier Baillin, Raluca Tiron, Didier Gasparutto, Christophe Brun, MINACOM, XLIM (XLIM), Université de Limoges (UNILIM)-Centre National de la Recherche Scientifique (CNRS)-Université de Limoges (UNILIM)-Centre National de la Recherche Scientifique (CNRS), Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)

Subjects
[PHYS]Physics [physics], Materials science, Fabrication, General Engineering, General Physics and Astronomy, Nanotechnology, 02 engineering and technology, Substrate (electronics), 010402 general chemistry, 021001 nanoscience & nanotechnology, 01 natural sciences, 0104 chemical sciences, law.invention, Etching (microfabrication), law, DNA nanotechnology, [CHIM]Chemical Sciences, DNA origami, General Materials Science, Process window, [PHYS.COND]Physics [physics]/Condensed Matter [cond-mat], Photolithography, 0210 nano-technology, Lithography, ComputingMilieux_MISCELLANEOUS
Abstract

DNA nanotechnology is currently widely explored and especially shows promises for advanced lithography due to its ability to define nanometer scale features. We demonstrate a 9 × 14 nm(2) hole pattern transfer from DNA origami into an SiO2 layer with a sub-10-nm resolution using anhydrous HF vapor in a semiconductor etching machine. We show that the resulting SiO2 pattern inherits its shape from the DNA structure within a process time ranging from 30 to 60 s at an etching rate of 0.2 nm/s. At 600 s of etching, the SiO2 pattern meets corrosion and the overall etching reaction is blocked. These results, in addition to the entire surface coverage by magnesium occurring on the substrate at a density of 1.1 × 10(15) atom/cm(2), define a process window, fabrication rules, and limits for DNA-based lithography.

Published
2016

22. Characterization of DNA ADP-ribosyltransferase activities of PARP2 and PARP3: new insights into DNA ADP-ribosylation

Author

Alexander A. Ishchenko, Bakhyt T. Matkarimov, M. M. Kutuzov, Ibtissam Talhaoui, Gabriella Zarkovic, Denis Biard, Ekaterina A. Belousova, Olga I. Lavrik, Didier Gasparutto, Christine Saint-Pierre, Stabilité Génétique et Oncogenèse (UMR 8200), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de Radiobiologie Cellulaire et Moléculaire (IRCM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Novosibirsk Institute of Chemical Biology and Fundamental Medecine, Intégrité du génome et cancers (IGC), Institut Gustave Roussy (IGR)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institute of Chemical Biology and Fundamental Medicine SB RAS, Institut Nanosciences et Cryogénie (INAC), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), National Laboratory Astana, Nazarbayev University [Kazakhstan], TEAM CELLULAR ENGINEERING AND HUMAN SYNDROMES, Commissariat à l’énergie atomique et aux énergies alternatives [Fontenay-aux-Roses] (CEA DSV), Institute of Chemical Biology and Fundamental Medicine [Novosibirsk, Russia] (ICBFM SB RAS), Siberian Branch of the Russian Academy of Sciences (SB RAS), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut Gustave Roussy (IGR)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Institut de Biologie François JACOB (JACOB)

Subjects
0301 basic medicine, genomic DNA, [SDV]Life Sciences [q-bio], Cell Cycle Proteins, DNA ADP-ribosylation, Genome Integrity, Repair and Replication, nicotinamide adenine dinucleotide adenosine diphosphate ribosyltransferase, Substrate Specificity, chemistry.chemical_compound, DNA Adducts, 0302 clinical medicine, A-DNA, DNA Breaks, Double-Stranded, Polymerase, ComputingMilieux_MISCELLANEOUS, mono adenosine diphosphate ribosylation, nicotinamide adenine dinucleotide adenosine diphosphate ribosyltransferase 3, bleomycin, biology, adenosine diphosphate, nicotinamide adenine dinucleotide adenosine diphosphate ribosyltransferase 2, HeLa cell line, unclassified drug, enzyme activity, priority journal, Biochemistry, 030220 oncology & carcinogenesis, glycosidase, enzyme active site, Poly(ADP-ribose) Polymerases, DNA modification, poly(adenosine diphosphate ribose), cell free system, DNA damage, Poly ADP ribose polymerase, DNA replication, Article, single stranded DNA break, Phosphates, 03 medical and health sciences, Genetics, double stranded DNA break, Humans, controlled study, enzyme mechanism, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, human, enzyme specificity, Adenosine Diphosphate Ribose, Oligonucleotide, human cell, DNA Breaks, molecular weight, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, DNA, 030104 developmental biology, chemistry, biology.protein, HeLa Cells
Abstract

International audience; Poly(ADP-ribose) polymerases (PARPs) act as DNA break sensors and catalyze the synthesis of polymers of ADP-ribose (PAR) covalently attached to acceptor proteins at DNA damage sites. It has been demonstrated that both mammalian PARP1 and PARP2 PARylate double-strand break termini in DNA oligonucleotide duplexes in vitro. Here, we show that mammalian PARP2 and PARP3 can PARylate and mono(ADP-ribosyl)ate (MARylate), respectively, 5-and 3-terminal phosphate residues at double-and single-strand break termini of a DNA molecule containing multiple strand breaks. PARP3catalyzed DNA MARylation can be considered a new type of reversible post-replicative DNA modification. According to DNA substrate specificity of PARP3 and PARP2, we propose a putative mechanistic model of PARP-catalyzed strand break-oriented ADP-ribosylation of DNA termini. Notably, PARPmediated DNA ADP-ribosylation can be more effective than PARPs' auto-ADP-ribosylation depending on the DNA substrates and reaction conditions used. Finally, we show an effective PARP3-or PARP2catalyzed ADP-ribosylation of high-molecular-weight (∼3-kb) DNA molecules, PARP-mediated DNA PARylation in cell-free extracts and a persisting signal of anti-PAR antibodies in a serially purified genomic DNA from bleomycin-treated poly(ADP-ribose) glycohydrolase-depleted HeLa cells. These results suggest that certain types of complex DNA breaks can be effectively ADP-ribosylated by PARPs in cellular response to DNA damage.

Published
2018

23. Tuning Optical Properties of Functionalized Gold Nanorods through Controlled Interactions with Organic Semiconductors

Author

Russell J. Composto, Didier Gasparutto, Guillaume Gines, Robert C. Ferrier, Patrice Rannou, Brigitte Pépin-Donat, University of Pennsylvania, Laboratoire Lésions des Acides Nucléiques (LAN), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Electronique Moléculaire Organique et Hybride (LEMOH), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), University of Pennsylvania [Philadelphia], Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Materials science, Photoluminescence, Performance, Wavelength, Au Nanorods, Nanoparticle, Nanotechnology, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Fluorescence, chemistry.chemical_compound, Rhodamine-B, Monolayer, Rhodamine B, Physical and Theoretical Chemistry, Cetrimonium bromide, Monolayers, [PHYS]Physics [physics], Distance, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, Organic semiconductor, Amphiphilic Triblock Copolymers, General Energy, Chemical engineering, chemistry, Nanoparticles, Probes, Nanorod, Absorption (chemistry), 0210 nano-technology
Abstract

International audience; A novel nanohybrid assembly consisting of gold nanorods (AuNRs) and organic semiconductors (OSCs) to tune optical properties is presented. OSCs are bound to specific areas of the AUNRs (e.g., end or side) by controlling the surface Chemistry of the AuNRs. AuNRs anisotropically fiinctionalized with cetrimonium bromide (CTAB) cysteine, poly(ethylene oxide) (PEO), and/or DNA. Rhodamine B (RhB) 5(6)-carboxyfluorescein (CF), or c-yanine3 (Cy3) are incubated with the anisotropically functionalized AuNRs. The resulting optical properties of the dispersed AuNRs are characterized via steady-state UVvis absorption and photoluminescence (PL) Spectroscopies as a function Of OSC concentration. We found that CF and RhB adsorb to the AuNR differently depending on the AuNR surface chemistry, which results in unique optical properties of the assembly. The distinctive spectra were used to dotermine the interaction between the OSC and the AuNIL Finally, we found that attaching Cy3 specifically to the ends of the AuNR via a DNA tether results in a larger emission quenching process than when Cy3 is not attached. This method of nanohybrid assembly can be extended to :other metal nanostructures and OSCs. Its generic nature qualifies it as new methodology to precisely assemble nano- and molecular objects with controlled (opto)electronic properties.

Published
2015

24. Redox Photocatalysis with Water-Soluble Core–Shell CdSe-ZnS Quantum Dots

Author

Jean-Marie Mouesca, Marina Gromova, Jérôme Chauvin, Serge Gambarelli, Didier Gasparutto, Timothée Chauviré, Vincent Maurel, Jean-Luc Ravanat, Colette Lebrun, Pierre-Henri Jouneau, Reconnaissance Ionique et Chimie de Coordination (RICC), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Magnetic Resonance (RM ), Modélisation et Exploration des Matériaux (MEM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire Lésions des Acides Nucléiques (LAN), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Etude des Matériaux par Microscopie Avancée (LEMMA ), Département de Chimie Moléculaire - Chimie Inorganique Redox Biomimétique (DCM - CIRE), Département de Chimie Moléculaire (DCM), Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), and Département de Chimie Moléculaire - Chimie Inorganique Redox (DCM - CIRE)

Subjects
Visible-Light Irradiation, One-Electron Oxidation, Inorganic chemistry, Semiconductor Nanocrystals, 02 engineering and technology, Reaction intermediate, 010402 general chemistry, Photochemistry, 01 natural sciences, Redox, Correlation-Energy, Nanorod Heterostructures, Core shell, chemistry.chemical_compound, Physical and Theoretical Chemistry, Charge Separation, Hydrogen production, [PHYS]Physics [physics], Singlet Oxygen, Chemistry, Singlet oxygen, Hydrogen-Production, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, Efficient, General Energy, Quantum dot, Hole Transfer, Photocatalysis, 0210 nano-technology, Visible spectrum
Abstract

International audience; The use of CdSe-ZnS type I quantum.clots as redox photocatalysts with visible light in water is investigated by monitoring the oxidation reaction of 8-oxo-2'-deoxyguanosine and the reduction of nitrophenylalanine derivatives. The detection of reaction intermediates and the identification and quantitation of reaction products establish that both reactions are photocatalyzed simultaneously by CdSe-ZnS type I quantum dots. The photocatalyzed reactions are observed without the need for sacrificial reactants and with high turnover numbers (>400). These results demonstrate that CdSe-ZnS type I quantum dots can work efficiently as redox photocatalysts in water solution.

Published
2015

25. Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2

Author

Kyung Duk Koh, Robert J. Crouch, Didier Gasparutto, Matilde Clarissa Malfatti, Christine Saint-Pierre, Sathya Balachander, Hyongi Chon, Gianluca Tell, Giulia Antoniali, Francesca Storici, Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, DNA Repair, RNase P, DNA repair, [SDV]Life Sciences [q-bio], Ribonuclease H, Endoribonuclease, Genome Integrity, Repair and Replication, Substrate Specificity, law.invention, 03 medical and health sciences, Endonuclease, chemistry.chemical_compound, law, DNA-(Apurinic or Apyrimidinic Site) Lyase, Genetics, Humans, [CHIM]Chemical Sciences, AP site, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Binding Sites, Models, Genetic, 030102 biochemistry & molecular biology, biology, DNA, Base excision repair, Ribonucleotides, Recombinant Proteins, Kinetics, 030104 developmental biology, Biochemistry, chemistry, biology.protein, Recombinant DNA, Oxidation-Reduction, HeLa Cells, Protein Binding
Abstract

Ribonucleoside 5′-monophosphates (rNMPs) are the most common non-standard nucleotides found in DNA of eukaryotic cells, with over 100 million rNMPs transiently incorporated in the mammalian genome per cell cycle. Human ribonuclease (RNase) H2 is the principal enzyme able to cleave rNMPs in DNA. Whether RNase H2 may process abasic or oxidized rNMPs incorporated in DNA is unknown. The base excision repair (BER) pathway is mainly responsible for repairing oxidized and abasic sites into DNA. Here we show that human RNase H2 is unable to process an abasic rNMP (rAP site) or a ribose 8oxoG (r8oxoG) site embedded in DNA. On the contrary, we found that recombinant purified human apurinic/apyrimidinic endonuclease-1 (APE1) and APE1 from human cell extracts efficiently process an rAP site in DNA and have weak endoribonuclease and 3′-exonuclease activities on r8oxoG substrate. Using biochemical assays, our results provide evidence of a human enzyme able to recognize and process abasic and oxidized ribonucleotides embedded in DNA.

Published
2017

26. Linear Chain Formation of Split-Aptamer Dimers on Surfaces Triggered by Adenosine

Author

Eric Peyrin, Chenze Lu, Didier Gasparutto, Arnaud Buhot, Yoann Roupioz, Christine Saint-Pierre, Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Département de pharmacochimie moléculaire, Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Adenosine, Polymers, Aptamer, Sequence (biology), Nanotechnology, 02 engineering and technology, Biosensing Techniques, 010402 general chemistry, 01 natural sciences, [SPI]Engineering Sciences [physics], Electrochemistry, medicine, General Materials Science, Surface plasmon resonance, [SPI.NANO]Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, Spectroscopy, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, Oligonucleotide, Chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Surfaces and Interfaces, Polymer, Aptamers, Nucleotide, Surface Plasmon Resonance, 021001 nanoscience & nanotechnology, Condensed Matter Physics, Small molecule, 0104 chemical sciences, Biophysics, 0210 nano-technology, Biosensor, medicine.drug
Abstract

The detection of small molecules impacts various fields; however, their small size and low concentration are usually the cause of limitations in their detection. Thus, the need for biosensors with appropriate probes and signal amplification strategies is required. Aptamers are appropriate probes selected specifically against small targets such as adenosine. The possibility to split aptamers in parts led to original amplification strategies based on sandwich assays. By combining the self-assembling of oligonucleotide dimers with split-aptamer dangling ends and a surface plasmon resonance imaging technique, we developed an original amplification approach based on linear chain formation in the presence of the adenosine target. In this article, on the basis of sequence engineering, we analyzed its performance and the effect of the probe grafting density on the length of the chains formed at the surface of the biosensor.

Published
2017

27. Metallic Conductive Nanowires Elaborated by PVD Metal Deposition on Suspended DNA Bundles

Author

Didier Gasparutto, Christophe Brun, Cheikh Tidiane-Diagne, Aurélie Thuaire, Raluca Tiron, Xavier Baillin, Pierre-Henri Elchinger, Guillaume Nonglaton, MINACOM, XLIM (XLIM), Université de Limoges (UNILIM)-Centre National de la Recherche Scientifique (CNRS)-Université de Limoges (UNILIM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie des Substances Naturelles (LCSN), Université de Limoges (UNILIM)-Génomique, Environnement, Immunité, Santé, Thérapeutique (GEIST FR CNRS 3503), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), French Alternative Energies and Atomic Energy Commission (A3DN), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)

Subjects
Materials science, Silicon, Nanowire, chemistry.chemical_element, Nanotechnology, 02 engineering and technology, Microscopy, Atomic Force, 010402 general chemistry, 01 natural sciences, Biomaterials, Microelectronics, [CHIM]Chemical Sciences, General Materials Science, Electrodes, Ohmic contact, Titanium, [PHYS]Physics [physics], Nanowires, business.industry, Drop (liquid), Electric Conductivity, DNA, General Chemistry, [CHIM.MATE]Chemical Sciences/Material chemistry, 021001 nanoscience & nanotechnology, 0104 chemical sciences, chemistry, Metals, Physical vapor deposition, Electrode, Optoelectronics, Gold, 0210 nano-technology, business, Biotechnology
Abstract

International audience; Metallic conductive nanowires (NWs) with DNA bundle core are achieved, thanks to an original process relying on double-stranded DNA alignment and physical vapor deposition (PVD) metallization steps involving a silicon substrate. First, bundles of DNA are suspended with a repeatable process between 2 aem high parallel electrodes with separating gaps ranging from 800 nm to 2 mu m. The process consists in the drop deposition of a DNA lambda-phage solution on the electrodes followed by a naturally evaporation step. The deposition process is controlled by the DNA concentration within the buffer solution, the drop volume, and the electrode hydrophobicity. The suspended bundles are finally metallized with various thicknesses of titanium and gold by a PVD e-beam evaporation process. The achieved NWs have a width ranging from a few nanometers up to 100 nm. The electrical behavior of the achieved 60 and 80 nm width metallic NWs is shown to be Ohmic and their intrinsic resistance is estimated according to different geometrical models of the NW section area. For the 80 nm width NWs, a resistance of about few ohms is established, opening exploration fields for applications in microelectronics.

Published
2017

28. Self-assembly of porphyrin–DNA hybrids into large flat nanostructures

Author

Stéphane Campidelli, Didier Gasparutto, Christine Saint-Pierre, Grégory Chatelain, Guillaume Clavé, Laboratoire Innovation en Chimie des Surfaces et NanoSciences (LICSEN UMR 3685), Nanosciences et Innovation pour les Matériaux, la Biomédecine et l'Energie (ex SIS2M) (NIMBE UMR 3685), Institut Rayonnement Matière de Saclay (IRAMIS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Rayonnement Matière de Saclay (IRAMIS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), CEA, Laboratoire Innovation en Chimie des Surfaces et NanoSciences (LICSEN), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut Rayonnement Matière de Saclay (IRAMIS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques [?-2019] (CREAB [?-2019]), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Institut Rayonnement Matière de Saclay (IRAMIS), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)

Subjects
Nanostructure, Porphyrins, 010405 organic chemistry, Oligonucleotide, Organic Chemistry, Context (language use), Nanotechnology, DNA, [CHIM.MATE]Chemical Sciences/Material chemistry, 010402 general chemistry, 01 natural sciences, Biochemistry, Porphyrin, 0104 chemical sciences, Nanostructures, chemistry.chemical_compound, chemistry, DNA nanotechnology, DNA origami, [CHIM]Chemical Sciences, Self-assembly, Physical and Theoretical Chemistry, ComputingMilieux_MISCELLANEOUS
Abstract

International audience; The main aim of nanotechnology is to create functional systems by controlling the matter at the nano-meter level. In this context DNA is a versatile building block for the fabrication of micrometer-scale objects with a subnanometer-scale resolution. Over the last 15 years, DNA nanotechnology has considerably developed with the invention of DNA origami, double crossover structures and molecule/ oligonucleotide hybrids. Our interest is focused on the combination of short complementary DNA sequences with organic molecules with a view to create large self-assembled nanostructures. Here we report on the synthesis of porphyrin derivatives bearing up to four 21-mer oligonucleotides and we demonstrate that the combination of the molecular hybrids containing complementary DNA strands leads to the formation of large nanostructures with micrometer-scale size.

Published
2017
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29. Anomalous electrophoretic migration of short oligodeoxynucleotides labelled with 5′-terminal Cy5 dyes

Author

Didier Gasparutto, Ghislaine Henneke, Tom Killelea, Céline Ralec, and Christine Saint-Pierre

Subjects
chemistry.chemical_classification, Exonuclease, 0303 health sciences, DNA clamp, biology, Oligonucleotide, Hybridization probe, Clinical Biochemistry, technology, industry, and agriculture, 010402 general chemistry, biology.organism_classification, 01 natural sciences, Biochemistry, 0104 chemical sciences, Analytical Chemistry, 03 medical and health sciences, chemistry.chemical_compound, chemistry, biology.protein, Nucleotide, Polymerase, Pyrococcus abyssi, DNA, 030304 developmental biology
Abstract

By using a fluorescent exonuclease assay, we reported unusual electrophoretic mobility of 5′-indocarbo-cyanine 5 (5′-Cy5) labelled DNA fragments in denaturing polyacrylamide gels. Incubation time and enzyme concentration were two parameters involved in the formation of 5′-Cy5-labelled degradation products, while the structure of the substrate was slightly interfering. Replacement of positively charged 5′-Cy5-labelled DNA oligonucleotides (DNA oligos) by electrically neutral 5′-carboxyfluorescein (5′-FAM) labelled DNA oligos abolished the anomalous migration pattern of degradation products. MS analysis demonstrated that anomalously migrating products were in fact 5′-labelled DNA fragments ranging from 1 to 8 nucleotides. Longer 5′-Cy5-labelled DNA fragments migrated at the expected position. Altogether, these data highlighted, for the first time, the influence of the mass/charge ratio of 5′-Cy5-labelled DNA oligos on their electrophoretic mobility. Although obtained by performing 3′ to 5′ exonuclease assays with the family B DNA polymerase from Pyrococcus abyssi, these observations represent a major concern in DNA technology involving most DNA degrading enzymes.

Published
2014

30. Tools and strategies for DNA damage interactome analysis

Author

Christophe Bounaix Morand du Puch, Ewa Barbier, Didier Gasparutto, Sylvie Sauvaigo, Jean Breton, Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), Laboratoire Lésions des Acides Nucléiques (LAN), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC)

Subjects
DNA Repair, DNA damage, DNA repair, [SDV]Life Sciences [q-bio], Health, Toxicology and Mutagenesis, Computational biology, Biology, Proteomics, Interactome, 03 medical and health sciences, 0302 clinical medicine, Genetics, Animals, Humans, [CHIM]Chemical Sciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein–DNA interaction, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Nuclear Proteins, Chromatin, ChIP-sequencing, 030220 oncology & carcinogenesis, Protein microarray, DNA Damage
Abstract

DNA is the target of multiple endogenous and exogenous agents generating chemical lesions on the double helix. Cellular DNA damage response pathways rely on a myriad of proteins interacting with DNA alterations. The cartography of this interactome currently includes well known actors of chromatin remodelling, DNA repair or proteins hijacked from their natural functions such as transcription factors. In order to go further into the characterisation of these protein networks, proteomics-based methods began to be used in the early 2000s. The strategies are diverse and include mainly (i) damaged DNA molecules used as targets on protein microarrays, (ii) damaged DNA probes used to trap within complex cellular extracts proteins that are then separated and identified by proteomics, (iii) identification of chromatin- bound proteins after a genotoxic stress, or (iv) identification of proteins associated with other proteins already known to be part of DNA damage interactome. All these approaches have already been performed to find new proteins recognizing oxidised bases, abasic sites, strand breaks or crosslinks generated by anticancer drugs such as nitrogen mustards and platinating agents. Identified interactions are generally confirmed using complementary methods such as electromobility shift assays or surface plasmon resonance. These strategies allowed, for example, demonstration of interactions between cisplatin-DNA crosslinks and PARP-1 or the protein complex PTW/PP. The next challenging step will be to understand the biological repercussions of these newly identified interactions which may help to unravel new mechanisms involved in genetic toxicology, discover new cellular responses to anticancer drugs or identify new biomarkers and therapeutic targets.

Published
2013

31. A high-throughput screen for detection of compound-dependent phosphodiester bond cleavage at abasic sites

Author

Marc C. Rideout, Nathalie Berthet, Benjamin Liet, Didier Gasparutto, Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Département de Chimie Moléculaire - Ingéniérie et Intéractions BioMoléculaires (DCM - I2BM), Département de Chimie Moléculaire (DCM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Stereochemistry, [SDV]Life Sciences [q-bio], Biophysics, Cleavage (embryo), Biochemistry, Chemical library, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Molecular beacon, [CHIM]Chemical Sciences, AP site, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Gel electrophoresis, Nuclease, Deoxyribonucleases, biology, Deoxyribose, DNA, Cell Biology, 3. Good health, 030104 developmental biology, chemistry, 030220 oncology & carcinogenesis, Phosphodiester bond, biology.protein
Abstract

We have employed a DNA molecular beacon with a real abasic site, namely a 2-deoxyribose, in a fluorescent high-throughput assay to identify artificial nucleases that cleave at abasic sites. We screened a 1280 compound chemical library and identified a compound that functions as an artificial nuclease. We validated a key structure-activity relationship necessary for abasic site cleavage using available analogs of the identified artificial nuclease. We also addressed the activity of the identified compound with dose titrations in the absence and presence of a source of non-specific DNA. Finally, we characterized the phosphodiester backbone cleavage at the abasic site using denaturing gel electrophoresis. This study provides a useful template for researchers seeking to rapidly identify new artificial nucleases.

Published
2016

32. Oxidative DNA damage and repair in the radioresistant archaeon Thermococcus gammatolerans

Author

Ewa Barbier, Christine Saint-Pierre, Aurore Gorlas, Amine Hachemi, Arnaud Lagorce, Murielle Dutertre, Lucie Morand, Jean Armengaud, Jean-Luc Ravanat, Thierry Douki, Jean Breton, Didier Gasparutto, Fabrice Confalonieri, Laboratoire Lésions des Acides Nucléiques (LAN), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Interactions Hôtes-Pathogènes-Environnements (IHPE), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Université de Perpignan Via Domitia (UPVD), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire Innovations technologiques pour la Détection et le Diagnostic (LI2D), Service de Pharmacologie et Immunoanalyse (SPI), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Médicaments et Technologies pour la Santé (MTS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Université de Perpignan Via Domitia (UPVD)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)

Subjects
0301 basic medicine, Proteomics, DNA Repair, Oxidative phosphorylation, Biology, Toxicology, Radiation Tolerance, Oxidative dna damage, 03 medical and health sciences, chemistry.chemical_compound, Radioresistance, Irradiation, 030102 biochemistry & molecular biology, [SDV.BID.EVO]Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE], Thermococcus gammatolerans, General Medicine, biology.organism_classification, Thermococcus, 030104 developmental biology, DNA, Archaeal, Biochemistry, chemistry, Transcriptome, Nucleoside, Oxidation-Reduction, DNA, Bacteria, DNA Damage
Abstract

International audience; The hyperthermophilic archaeon Thermococcus gammatolerans can resist huge doses of γ-irradiation, up to 5.0 kGy, without loss of viability. The potential to withstand such harsh conditions is probably due to complementary passive and active mechanisms, including repair of damaged chromosomes. In this work, we documented the formation and repair of oxidative DNA lesions in T. gammatolerans. The basal level of the oxidized nucleoside, 8-oxo-2′-deoxyguanosine (8-oxo-dGuo), was established at 9.2 (± 0.9) 8-oxo-dGuo per 106 nucleosides, a higher level than those usually measured in eukaryotic cells or bacteria. A significant increase in oxidative damage, i.e., up to 24.2 (± 8.0) 8-oxo-dGuo/106 nucleosides, was measured for T. gammatolerans exposed to a 5.0 kGy dose of γ-rays. Surprisingly, the yield of radiation-induced modifications was lower than those previously observed for human cells exposed to doses corresponding to a few grays. One hour after irradiation, 8-oxo-dGuo levels were significantly reduced, indicating an efficient repair. Two putative base excision repair (BER) enzymes, TGAM_1277 and TGAM_1653, were demonstrated both by proteomics and transcriptomics to be present in the cells without exposure to ionizing radiation. Their transcripts were moderately upregulated after gamma irradiation. After heterologous production and purification of these enzymes, biochemical assays based on electrophoresis and MALDI-TOF (matrix-assisted laser desorption ionization–time of flight) mass spectrometry indicated that both have a β-elimination cleavage activity. TGAM_1653 repairs 8-oxo-dGuo, whereas TGAM_1277 is also able to remove lesions affecting pyrimidines (1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd)). This work showed that in normal growth conditions or in the presence of a strong oxidative stress, T. gammatolerans has the potential to rapidly reduce the extent of DNA oxidation, with at least these two BER enzymes as bodyguards with distinct substrate ranges.

Published
2016

33. Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro

Author

M. M. Kutuzov, Maria V. Sukhanova, N. A. Lebedeva, Christine Saint-Pierre, Olga I. Lavrik, Alexander A. Ishchenko, Murat Saparbaev, Didier Gasparutto, Gabriella Zarkovic, Bakhyt T. Matkarimov, Ibtissam Talhaoui, Stabilité Génétique et Oncogenèse (UMR 8200), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institute of Chemical Biology and Fundamental Medicine [Novosibirsk, Russia] (ICBFM SB RAS), Siberian Branch of the Russian Academy of Sciences (SB RAS), Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Structures et propriétés d'architectures moléculaire (SPRAM - UMR 5819), Institut Nanosciences et Cryogénie (INAC), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), National Laboratory Astana, Nazarbayev University [Kazakhstan], Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk State University (NSU), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, double stranded DNA, [SDV]Life Sciences [q-bio], Poly (ADP-Ribose) Polymerase-1, protein binding, Genome Integrity, Repair and Replication, Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide adenosine diphosphate ribosyltransferase, Substrate Specificity, chemistry.chemical_compound, DNA Adducts, Mice, 0302 clinical medicine, adenosine diphosphate ribosylation, molecular biology, animal, genetics, DNA Breaks, Double-Stranded, chemical bond, ComputingMilieux_MISCELLANEOUS, Polymerase, mass spectrometry, DNA cleavage, DNA strand breakage, Hydrolysis, nicotinamide adenine dinucleotide adenosine diphosphate ribosyltransferase 1, nicotinamide adenine dinucleotide adenosine diphosphate ribosyltransferase 2, monomer, structure analysis, unclassified drug, enzyme activity, Biochemistry, priority journal, 030220 oncology & carcinogenesis, glycosidase, Poly(ADP-ribose) Polymerases, DNA modification, Protein Binding, oligonucleotide, in vitro study, DNA repair, poly(ADP ribose)glycosidase, Poly ADP ribose polymerase, Biology, Article, Catalysis, 03 medical and health sciences, Deoxyribonucleotide, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Genetics, [CHIM]Chemical Sciences, biochemistry, double stranded DNA break, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, controlled study, human, enzyme specificity, nicotinamide adenine dinucleotide, mouse, phosphate, cordycepin, Oligonucleotide, DNA fragment, nucleotide sequence, DNA, NAD, 030104 developmental biology, chemistry, Parp2 protein, mouse, Phosphodiester bond, DNA adduct, biology.protein, metabolism
Abstract

International audience; Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD +) to catalyse the synthesis of a long branched poly(ADPribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single-and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3-cordycepin, 5-and 3phosphate and also to 5-phosphate of a singlestranded oligonucleotide. PARP2 preferentially ADPribosylated the nicked/gapped DNA duplexes containing 5-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2 ,1-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1 of ADPribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs.

Published
2016

34. A DNA array based on clickable lesion-containing hairpin probes for multiplexed detection of base excision repair activities

Author

Guillaume Nonglaton, Christine Saint-Pierre, Mélanie Flaender, Didier Gasparutto, Guillaume Costa, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), [GIN] Grenoble Institut des Neurosciences (GIN), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)

Subjects
0301 basic medicine, DNA Repair, DNA repair, [SDV]Life Sciences [q-bio], Biosensing Techniques, Biochemistry, Analytical Chemistry, 03 medical and health sciences, chemistry.chemical_compound, Methoxyamine, DNA-(Apurinic or Apyrimidinic Site) Lyase, Electrochemistry, Humans, Environmental Chemistry, [CHIM]Chemical Sciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Uracil-DNA Glycosidase, Spectroscopy, ComputingMilieux_MISCELLANEOUS, Oligonucleotide Array Sequence Analysis, Hybridization probe, Uracil, DNA, Base excision repair, Molecular biology, 030104 developmental biology, chemistry, DNA glycosylase, Click Chemistry, DNA microarray, DNA Probes, HeLa Cells
Abstract

DNA is under continuous assault by environmental and endogenous reactive oxygen and alkylating species, inducing the formation of mutagenic, toxic and genome destabilizing nucleobase lesions. Due to the implications of such genetic alterations in cell death, aging, inflammation, neurodegenerative diseases and cancer, many efforts have been devoted to developing assays that aim at analyzing DNA repair activities from purified enzymes or cell extracts. The present work deals with the conception and application of a new, miniaturized and parallelized on surface-DNA biosensor to measure base excision repair (BER) activities. Such a bio-analytical tool was built by using the "click chemistry" approach to immobilize, on a glass slide, fluorescent stem-loop DNA probes, which contain a specific nucleobase lesion. The performance of this new high-throughput DNA repair analysis technology was determined by detecting uracil N-glycosylase and AP-endonuclease activities from purified enzymes or in cell extracts. The applications of this device were extended to analyze, in cell extracts, the ability of two inhibitors (Uracil glycosylase inhibitor (Ugi) and methoxyamine (MX)) to block the excision of uracil and the cleavage of AP sites, respectively. Altogether, our results show that this new fluorescent DNA microarray platform provides an easy, rapid and robust method for detecting DNA N-glycosylase and AP-endonuclease activities and evaluating the effects of BER inhibitors in a multiplexed fashion.

Published
2016

35. The formation of double-strand breaks at multiply damaged sites is driven by the kinetics of excision/incision at base damage in eukaryotic cells

Author

Evelyne Sage, Anne Reynaud-Angelin, Didier Gasparutto, Stanislav G. Kozmin, and Yuliya Sedletska

Subjects
DNA Repair, DNA damage, DNA repair, Genetic Vectors, Cell, Saccharomyces cerevisiae, Genome Integrity, Repair and Replication, chemistry.chemical_compound, Transformation, Genetic, Plasmid, hemic and lymphatic diseases, Genetics, medicine, DNA Breaks, Double-Stranded, DNA Cleavage, biology, Oligonucleotide, biology.organism_classification, Molecular biology, Yeast, Kinetics, medicine.anatomical_structure, chemistry, DNA, DNA Damage
Abstract

It has been stipulated that repair of clustered DNA lesions may be compromised, possibly leading to the formation of double-strand breaks (DSB) and, thus, to deleterious events. Using a variety of model multiply damaged sites (MDS), we investigated parameters that govern the formation of DSB during the processing of MDS. Duplexes carrying MDS were inserted into replicative or integrative vectors, and used to transform yeast Saccharomyces cerevisiae. Formation of DSB was assessed by a relevant plasmid survival assay. Kinetics of excision/incision and DSB formation at MDS was explored using yeast cell extracts. We show that MDS composed of two uracils or abasic sites, were rapidly incised and readily converted into DSB in yeast cells. In marked contrast, none of the MDS carrying opposed oG and hU separated by 3–8 bp gave rise to DSB, despite the fact that some of them contained preexisting single-strand break (a 1-nt gap). Interestingly, the absence of DSB formation in this case correlated with slow excision/incision rates of lesions. We propose that the kinetics of the initial repair steps at MDS is a major parameter that direct towards the conversion of MDS into DSB. Data provides clues to the biological consequences of MDS in eukaryotic cells.

Published
2009

36. Absence of 2′-deoxyguanosine-carbon 8-bound ochratoxin A adduct in rat kidney DNA monitored by isotope dilution LC-MS/MS

Author

Janique Richoz, Sibel Ozden, Angela Mally, Wolfgang Dekant, Christophe Cavin, Maricel Marin-Kuan, Benoît Schilter, Heiko Ihmels, Daniela Otto, Thierry Delatour, and Didier Gasparutto

Subjects
Male, Models, Molecular, Ochratoxin A, Molecular Conformation, Administration, Oral, Isotope dilution, Kidney, Mass Spectrometry, Adduct, chemistry.chemical_compound, DNA adduct, Animals, Deoxyguanosine, Ochratoxin, Chromatography, Nucleotides, Oligonucleotide, DNA, Mycotoxins, Ochratoxins, Rats, Inbred F344, Rats, chemistry, Chromatography, Liquid, Food Science, Biotechnology
Abstract

The contribution of DNA adduct formation in the carcinogenic action of the mycotoxin ochratoxin A (OTA) has been subject to much debate. Recently, a carbon-bonded ochratoxin A-2'-deoxyguanosine adduct (dGuoOTA) formed by photochemical reaction in vitro has been shown by 32 P-postlabeling/ TLC to comigrate with a spot detected in DNA isolated from rat and pig kidney following exposure to OTA. Considering the large body of evidence arguing against covalent DNA binding of OTA and the poor resolution and specificity of postlabeling analysis, we developed a stable isotope dilution LC-MS/MS method to analyze dGuoOTA in kidney DNA isolated from rats treated with OTA. dGuoOTA and nitrogen-15-labeled dGuoOTA ( 15 N 5 -dGuoOTA) were prepared by photoirradiation of OTA in the presence of dGuo or nitrogen-15-labeled dGuo. Conditions for DNA hydrolysis were optimized using a synthetic oligonucleotide containing dGuoOTA to ensure complete release of dGuoOTA. The LOD of the method (S/N > 3) was 10 fmol dGuoOTA on-column. However, dGuoOTA was not detected in DNA samples isolated from male F344 rats treated with OTA for up to 90 days at doses known to cause renal tumor formation. Detection limits, calculated for each individual sample based on the absolute LOD and the amount of DNA injected, were as low as 3.5 dGuoOTA/10 9 nucleotides. These data are consistent with previous results showing lack of DNA adduct formation by OTA and demonstrate that dGuoOTA is not formed in biologically relevant amounts under physiological conditions in vivo.

Published
2008

37. Characterization of the DNA repair spore photoproduct lyase enzyme from Clostridium acetobutylicum: A radical-SAM enzyme

Author

Mohamed G. Atta, Sandrine Ollagnier-de-Choudens, Alexia Chandor, Yiannis Sanakis, Didier Gasparutto, Marc Fontecave, Serge Gambarelli, Yvain Nicolet, Thierry Douki, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Lésions des Acides Nucléiques (LAN), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institute of Materials Science [Athens], National Center for Scientific Research 'Demokritos' (NCSR), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)

Subjects
Clostridium acetobutylicum, General Chemical Engineering, Bacillus subtilis, [CHIM.INOR]Chemical Sciences/Inorganic chemistry, 010402 general chemistry, medicine.disease_cause, 01 natural sciences, 03 medical and health sciences, medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Escherichia coli, ComputingMilieux_MISCELLANEOUS, Spore photoproduct lyase, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, General Chemistry, biology.organism_classification, 0104 chemical sciences, Enzyme, Ribonucleotide reductase, chemistry, Biochemistry, sense organs, Bacterial spore, Radical SAM
Abstract

Spore photoproduct lyase (SPL) is a “Radical-SAM” repair enzyme which catalyzes the cleavage of spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine), a specific lesion found in bacterial spore DNA, to thymine monomers by a free-radical mechanism. The enzyme requires S-adenosyl- l -methionine (SAM) and a [4Fe–4S] cluster for activity. SPL from Bacillus subtilis has been difficult to isolate and characterize due to problems with the solubility and stability of the overexpressed protein in Escherichia coli and the lability of the [Fe–S] cluster, even if the protein was purified under strictly anaerobic conditions. In order to overcome these problems we searched for another SPL enzyme and we found that the recombinant SPL enzyme from Clostridium acetobutylicum, isolated either aerobically or anaerobically from overexpressing E. coli, behaves more stably than the B. subtilis one. We report here a complete spectroscopic and biochemical characterization of this enzyme. In particular we show for the first time that, using HYSCORE spectroscopy, SAM binds to the cluster as observed in the case of other members of the “Radical-SAM” enzyme family such as the activases of pyruvate formate lyase and ribonucleotide reductase.

Published
2007

38. Development and Application of a Novel Immunoassay for Measuring Oxidative DNA Damage in the Environment¶

Author

David L. Mitchell, Didier Gasparutto, Jarah A. Meador, Laxmi Paniker, Wade H. Jeffrey, and Jean Cadet

Subjects
Radioimmunoassay, Pyrimidine dimer, Biochemistry, Adduct, Cell Line, chemistry.chemical_compound, medicine, Electrochemistry, Animals, Humans, Bovine serum albumin, Physical and Theoretical Chemistry, Chromatography, High Pressure Liquid, Detection limit, Chromatography, medicine.diagnostic_test, biology, General Medicine, Ligand (biochemistry), Molecular biology, Oxidative Stress, chemistry, Immunoassay, biology.protein, Sunlight, Cattle, Hapten, DNA, DNA Damage, Environmental Monitoring
Abstract

We developed a facile, cost-effective competitive binding assay for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in DNA, using a polyclonal rabbit antiserum raised against an 8-oxodGuo hapten coupled to bovine serum albumin and radiolabeled synthetic ligand containing multiple 8-oxodGuo residues. This radioimmunoassay (RIA) displays a high affinity for 8-oxodGuo in DNA, with a detection limit of approximately 1 adduct in 10(5) bases of DNA. 8-oxodGuo standards for RIA were quantified by high-performance liquid chromatography and electrochemical detection in DNA diluted in methylene blue and exposed to visible light. As an initial application we quantified 8-oxodGuo in dosimeters deployed at increasing depths in the Southern Ocean during the austral spring of the 1998 field season or at the surface at Palmer Station, Antarctica, throughout the 1999 field season. Cyclobutane pyrimidine dimers (CPD) were quantified using an established RIA. We found that the frequency of both photoproducts decreased with depth. However, CPD induction was attenuated at a faster rate than 8-oxodGuo, correlating with the differential attenuation of solar ultraviolet wavelengths in the water column. CPD induction was closely related with ultraviolet-B radiation (UVB) attenuation, whereas the lower attenuation of 8-oxodGuo suggests that oxidative damage is more closely related to ultraviolet-A radiation (UVA) irradiance. The ratio of 8-oxodGuo: CPD was also found to covary with changes in stratospheric ozone concentrations at Palmer Station. These data demonstrate the usefulness of these assays for environmental photobiology and the potential for their use in studying the relative impacts of UVB versus UVA, including ozone depletion events.

Published
2007

39. Synthesis of a multi-branched porphyrin-oligonucleotide scaffold for the construction of DNA-Based nano-architecture

Author

Didier Gasparutto, Grégory Chatelain, Olivier Piétrement, Stéphane Campidelli, Guillaume Clavé, Eric Le Cam, Laboratoire Innovation en Chimie des Surfaces et NanoSciences (LICSEN UMR 3685), Nanosciences et Innovation pour les Matériaux, la Biomédecine et l'Energie (ex SIS2M) (NIMBE UMR 3685), Institut Rayonnement Matière de Saclay (IRAMIS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Rayonnement Matière de Saclay (IRAMIS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Lésions des Acides Nucléiques (LAN), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), ANR-09-NANO-0005,f-DNA,Matériaux Fonctionnels à Base d'Assemblages Fullerene/ADN Bi- et Tridimensionnels(2009), Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Innovation en Chimie des Surfaces et NanoSciences (LICSEN), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Institut Rayonnement Matière de Saclay (IRAMIS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Centre National de la Recherche Scientifique (CNRS)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
chemistry.chemical_compound, Scaffold, chemistry, Oligonucleotide, law, Colloidal gold, [CHIM.MATE]Chemical Sciences/Material chemistry, Electron microscope, Porphyrin, Combinatorial chemistry, DNA, ComputingMilieux_MISCELLANEOUS, law.invention
Abstract

International audience; Porphytin-oligonudeotide hybrids containing one to four DNA strands (P1-P4) have been synthesised and fully characterised. The hybrid P4, which inserts one porphyrin and four DNA fragments, was combined with gold nanoparticles and images by electron microscopy.

Published
2015

40. Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways

Author

Zhiger Akishev, Zhi Liu, Christine Saint-Pierre, Nicholas E. Geacintov, Bakhyt T. Matkarimov, Ibtissam Talhaoui, Murat Saparbaev, Vladimir Shafirovich, Didier Gasparutto, Stabilité Génétique et Oncogenèse (UMR 8200), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), New York University [New York] (NYU), NYU System (NYU), School of Communication and Information Engineering [Shanghai], Shanghai University, Chimie pour la Reconnaissance et l’Etude d’Assemblages Biologiques (CREAB), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Structures et propriétés d'architectures moléculaire (SPRAM - UMR 5819), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Al-Farabi Kazakh National University [Almaty] (KazNU), National Laboratory Astana, Nazarbayev University [Kazakhstan], Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Al-Farabi Kazakh National University, Institut Gustave Roussy (IGR), Laboratoire Lésions des Acides Nucléiques (LAN), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), and Department of Molecular Biology and Genetics

Subjects
inorganic chemicals, DNA Repair, UVRABC nuclease, Guanine, DNA damage, DNA repair, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Oligonucleotides, DNA and Chromosomes, Radical-Induced Formation, Biochemistry, DNA Glycosylases, chemistry.chemical_compound, Humans, AP site, Molecular Biology, [PHYS]Physics [physics], Base Sequence, DNA, Cell Biology, Base excision repair, Molecular biology, Human-Cells, Mammalian-Cells, chemistry, DNA glycosylase, Oxidation-Reduction, Escherichia-Coli, HeLa Cells, Nucleotide excision repair
Abstract

International audience; Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O$_2$. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) $Nucleic$ $Acids$ $Res.$ 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3'-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins.

Published
2015

41. A New Tool for NMR Crystallography: Complete C-13/N-15 Assignment of Organic Molecules at Natural Isotopic Abundance Using DNP-Enhanced Solid-State NMR

Author

Katharina Märker, Morgane Pingret, Sabine Hediger, Jean-Marie Mouesca, Didier Gasparutto, Gaël De Paëpe, Magnetic Resonance (RM ), Modélisation et Exploration des Matériaux (MEM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire Lésions des Acides Nucléiques (LAN), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), ANR-12-BS08-0016,CryoMAS4DNP,Polarisation dynamique nucléaire en rotation à l'angle magique à très basse température et à haut champ magnétique(2012), ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
[PHYS]Physics [physics], 010405 organic chemistry, Chemistry, Chemical shift, Resolution (electron density), Crystal-Structure Prediction, Natural abundance, General Chemistry, Fluorine-19 NMR, Nuclear magnetic resonance crystallography, Guanosine Derivatives, 010402 general chemistry, 01 natural sciences, Biochemistry, Catalysis, 0104 chemical sciences, Crystallography, Colloid and Surface Chemistry, Solid-state nuclear magnetic resonance, Dynamic nuclear-polarization, Molecule, C-13-C-13 correlation spectroscopy, Phosphorus-31 NMR spectroscopy
Abstract

International audience; NMR crystallography of organic molecules at natural isotopic abundance (NA) strongly relies on the comparison of assigned experimental and computed NMR chemical shifts. However, a broad applicability of this approach is often hampered by the still limited H-1 resolution and/or difficulties in assigning C-13 and N-15 resonances without the use of structure-based chemical shift calculations. As shown here, such difficulties can be overcome by C-13-C-13 and for the first time N-15-C-13 correlation experiments, recorded with the help of dynamic nuclear polarization. We present the complete de novo C-13 and N-15 resonance assignment at NA of a self-assembled 2'-deoxyguanosine derivative presenting two different molecules in the asymmetric crystallographic unit cell. This de novo assignment method is exclusively based on aforementioned correlation spectra and is an important addition to the NMR crystallography approach, rendering firstly H-1 assignment straightforward, and being secondly a prerequisite for distance measurements with solid-state NMR.

Published
2015

42. Modification of Guanine with PhotolabileN-Hydroxypyridine-2(1H)-thione: Monomer Synthesis, Oligonucleotide Elaboration, and Photochemical Studies

Author

Despoina Vrantza, Jean Cadet, Susana Encinas, Panagiotis Kaloudis, Michael Orfanopoulos, Miguel A. Miranda, Didier Gasparutto, Cecilia Paris, Leondios Leondiadis, Thanasis Gimisis, and Georgios C. Vougioukalakis

Subjects
Oligonucleotide, Guanine, Organic Chemistry, Photodissociation, Guanosine, Oligonucleotide synthesis, Photochemistry, Biochemistry, Catalysis, Inorganic Chemistry, chemistry.chemical_compound, Monomer, chemistry, Sulfanyl, Drug Discovery, Physical and Theoretical Chemistry, DNA
Abstract

The photochemistry of N-hydroxypyridine-2(1H)-thione (NHPT), inserted as a photolabile modifier at the 6-position of 2′-deoxyguanosine or guanosine, has been evaluated. In particular, 6-[(1-oxidopyridin-2-yl)sulfanyl]- (1a) and 6-[(pyridin-2-yl)sulfanyl]-2′,6-dideoxyguanosine (2a), novel photolabile derivatives of the natural nucleosides, were synthesized and characterized. The observed photolysis products of 1a in organic solvents could only be rationalized by assuming a rapid equilibrium with the corresponding 6-[(2-thioxopyridin-1(2H)-yl)oxy] analogue 3a (Scheme 5). Transient spectroscopy of 1a indicated a strong triplet-excited state suitable for triplet triplet energy transfer or singlet-oxygen generation. The NHPT function was stable enough for (slightly modified) automated solid-phase oligonucleotide synthesis. The utility of the above compounds is discussed, as well as their potential use in photosensitization of reactive oxygen species in DNA.

Published
2006

43. Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs

Author

Didier Gasparutto, Jack Ballantyne, John P. McDonald, Ashley Hall, Roger Woodgate, and Jean Cadet

Subjects
DNA polymerase, Archaeal Proteins, Molecular Sequence Data, ved/biology.organism_classification_rank.species, Polymerase Chain Reaction, Article, Genes, Archaeal, law.invention, chemistry.chemical_compound, law, Enzyme Stability, Genetics, Humans, Cloning, Molecular, DNA Polymerase beta, Phylogeny, Polymerase chain reaction, Sulfolobus shibatae, Base Sequence, biology, ved/biology, Temperature, Multiple displacement amplification, Paleontology, DNA, Templates, Genetic, Amplicon, Archaea, DNA Fingerprinting, Molecular biology, chemistry, DNA profiling, Sulfolobaceae, biology.protein, TOPO cloning, K562 Cells, Taq polymerase, DNA Damage
Abstract

For many years, Taq polymerase has served as the stalwart enzyme in the PCR amplification of DNA. However, a major limitation of Taq is its inability to amplify damaged DNA, thereby restricting its usefulness in forensic applications. In contrast, Y-family DNA polymerases, such as Dpo4 from Sulfolobus solfataricus, can traverse a wide variety of DNA lesions. Here, we report the identification and characterization of five novel thermostable Dpo4-like enzymes from Acidianus infernus, Sulfolobus shibatae, Sulfolobus tengchongensis, Stygiolobus azoricus and Sulfurisphaera ohwakuensis, as well as two recombinant chimeras that have enhanced enzymatic properties compared with the naturally occurring polymerases. The Dpo4-like polymerases are moderately processive, can substitute for Taq in PCR and can bypass DNA lesions that normally block Taq. Such properties make the Dpo4-like enzymes ideally suited for the PCR amplification of damaged DNA samples. Indeed, by using a blend of Taq and Dpo4-like enzymes, we obtained a PCR amplicon from ultraviolet-irradiated DNA that was largely unamplifyable with Taq alone. The inclusion of thermostable Dpo4-like polymerases in PCRs, therefore, augments the recovery and analysis of lesion-containing DNA samples, such as those commonly found in forensic or ancient DNA molecular applications.

Published
2006

44. Synthesis of a Convenient Thymidine Glycol Phosphoramidite Monomer and Its Site-specific Incorporation into DNA Fragments

Author

Didier Gasparutto, Solveig Roussel, Jean Cadet, and Sonia Cognet

Subjects
Phosphoramidite, DNA damage, Lability, Diol, General Medicine, Biochemistry, Combinatorial chemistry, Chemical synthesis, Nucleobase, chemistry.chemical_compound, Organophosphorus Compounds, Monomer, Oligodeoxyribonucleotides, chemistry, Genetics, Molecular Medicine, Organic chemistry, Oxidation-Reduction, DNA, DNA Damage, Thymidine
Abstract

An original phosphoramidite building block of the thymidine glycol lesion has been prepared taking into account the additional diol function and the high lability of this oxidatively induced nucleobase damage. Then the modified nucleoside was site-specifically inserted into DNA fragments by solid support assembling followed by a "one-step" mild final deprotection treatment.

Published
2005

45. EPOXIDE ADDUCTS AT THE GUANINE RESIDUE WITHIN SINGLE-STRANDED DNA CHAINS: REACTIVITY AND STABILITY STUDIES

Author

Christine Saint-Pierre, Jean Cadet, T. Ramirez-Fuentes, Didier Gasparutto, and T. Michel

Subjects
Guanine, Stereochemistry, DNA damage, Oligonucleotides, DNA, Single-Stranded, Epoxide, Biochemistry, Nucleobase, DNA Adducts, chemistry.chemical_compound, Residue (chemistry), Genetics, Chromatography, High Pressure Liquid, Oligonucleotide, DNA, General Medicine, Models, Chemical, chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Butanes, Epoxy Compounds, Nucleic Acid Conformation, Molecular Medicine, Electrophoresis, Polyacrylamide Gel, Chemical stability, DNA Damage
Abstract

Emphasis was placed in this work on the assessment of structural and biological features of nucleobase adducts that result from the reaction of DNA with epoxide derivatives. Thus we have prepared and characterized a set of site-specifically modified oligonucleotides at N7-position of a guanine residue, upon reaction with diepoxibutane, with the purpose of further investigating some of their biochemical features. The stability of the lesion-containing DNA fragments has also been investigated and clearly shows that the latter modified oligomers may be used as substrates for in vitro enzymatic assays, aimed at determining the biological effects within cell of these chemically induced DNA damage.

Published
2005

46. Processing of a complex multiply damaged DNA site by human cell extracts and purified repair proteins

Author

Evelyne Sage, Grégory Eot-Houllier, Didier Gasparutto, Séverine Eon-Marchais, Génotoxicologie et cycle cellulaire (GCC), Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)

Subjects
Cell Extracts, Guanine, DNA Repair, DNA damage, DNA repair, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Biology, Cleavage (embryo), Article, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Radiation, Ionizing, Genetics, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Uracil, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Cell Line, Transformed, [SDV.GEN]Life Sciences [q-bio]/Genetics, 0303 health sciences, Bacteria, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Base excision repair, DNA repair protein XRCC4, Molecular biology, DNA Repair Enzymes, chemistry, DNA glycosylase, 030220 oncology & carcinogenesis, DNA, Nucleotide excision repair, DNA Damage
Abstract

Clustered DNA lesions, possibly induced by ionizing radiation, constitute a trial for repair processes. Indeed, recent studies suggest that repair of such lesions may be compromised, potentially leading to the formation of lethal double-strand breaks (DSBs). A complex multiply damaged site (MDS) composed of 8-oxoguanine and 8-oxoadenine on one strand, 5-hydroxyuracil, 5-formyluracil and a 1 nt gap on the other strand, within 17 bp was built and used to challenge several steps of base excision repair (BER) pathway with human whole-cell extracts and purified repair enzymes as well. We show a hierarchy in the processing of lesions within the MDS, in particular at the base excision step. In the present configuration, efficient excision of 5-hydroxyuracil and low cleavage at 8-oxoguanine prevent DSB formation and generate a short single-stranded region carrying the 8-oxoguanine. On the other hand, rejoining of the 1 nt gap occurs by the short-patch BER pathway, but is slightly retarded by the presence of the oxidized bases. Taken together, our results suggest a hierarchy in the processing of the lesions within the MDS, which prevents the formation of DSB, but would dramatically enhance mutagenesis. They also indicate that the mutagenic (or lethal) consequences of a complex MDS will largely depend on the first event in the processing of the MDS.

Published
2005

47. Radiation-Induced DNA Damage: Formation, Measurement, and Biochemical Features

Author

Sylvie Sauvaigo, Jean Cadet, Thierry Douki, Didier Gasparutto, Evelyne Muller, Jean-Pierre Pouget, Anthony Romieu, Sophie Bellon, Jean-Luc Ravanat, and Sandrine Frelon

Subjects
DNA Repair, Chemistry, DNA damage, DNA repair, Guanine, Health, Toxicology and Mutagenesis, Free radical damage to DNA, DNA, General Medicine, Toxicology, Molecular biology, Substrate Specificity, Pathology and Forensic Medicine, Thymine, Comet assay, chemistry.chemical_compound, DNA Repair Enzymes, Biochemistry, Gamma Rays, DNA glycosylase, Mutagenesis, Site-Directed, Animals, Humans, Radiation Induced DNA Damage, DNA Damage
Abstract

Most of the reactions induced by *OH radicals (indirect effects) and by one-electron oxidation (direct effects) as the result of exposure to ionizing radiation may be described for the four main DNA nucleobases. Relevant information is now available on the formation of single and tandem base lesions implicating guanine as the most susceptible DNA component to the deleterious effects of ionizing radiation. In contrast, there is still a paucity of information on the radiation-induced formation of base damage within cellular DNA. This is mostly a result of difficulties associated with the measurement of oxidized purine and pyrimidine bases that appear to be generated in very low yields. This is illustrated by the measurement of low amounts of E. coli formamidopyrimidine glycosylase- and endonuclease-III-sensitive sites in the DNA of neoplastic monocytes upon exposure to gamma rays (48 and 53 per 10(9) bases and per Gy, respectively) using a modified comet assay (the overall number of strand breaks and alkali-labile sites was estimated to be 130 per 10(9) bases and per Gy). More specifically, the level of several radiation-induced modified bases, including thymine glycols, 5-formyluracil, 5-(hydroxymethyl)uracil, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroadenine, together with related formamidopyrimidine derivatives was assessed using the suitable HPLC-MS/MS method. Information is also provided on the substrate specificity of DNA repair enzymes and the mutagenic potential of base lesions using site-specific modified oligonucleotides as the probes.

Published
2004

48. DNA for Nanopackaging: A Promising Bottom-Up Approach

Author

Cheikh Tidiane Diagne, Raluca Tiron, Xavier Baillin, Pierre-Henri Elchinger, Aurélie Thuaire, Didier Gasparutto, Christophe Brun, and Simona Torrengo

Subjects
Nanostructure, Materials science, business.industry, Mechanical Engineering, Nanowire, Potential candidate, Nanotechnology, 02 engineering and technology, 010402 general chemistry, 021001 nanoscience & nanotechnology, 01 natural sciences, Biological materials, 0104 chemical sciences, Miniaturization, Microelectronics, Electrical and Electronic Engineering, 0210 nano-technology, business, Nanoscopic scale
Abstract

Deoxyribonucleic acid (DNA) is presented as a potential candidate to develop nanopackaging solutions in the field of microelectronics by a bottom-up approach. Such an approach could overcome the miniaturization limitations of the current top-down approaches. By its particular and unique properties of self-assembling and the capability to be functionalized, DNA is a versatile biological material that can be used in many different ways to build multiscale nanostructures and especially conductive nanowires (NWs). These NWs could be implemented as potential future nanoscale interconnections.

Published
2017

49. Ethylene oxide and propylene oxide derived N7-alkylguanine adducts are bypassed accurately in vivo

Author

Jean Cadet, Robert P. Fuchs, Gerard Mazón, Gaëlle Philippin, Didier Gasparutto, Laboratoire Pierre Aigrain (LPA), Fédération de recherche du Département de physique de l'Ecole Normale Supérieure - ENS Paris (FRDPENS), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Lésions des Acides Nucléiques (LAN), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, University of Manchester [Manchester], Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Diderot - Paris 7 (UPD7)-Fédération de recherche du Département de physique de l'Ecole Normale Supérieure - ENS Paris (FRDPENS), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and Gasparutto, Didier

Subjects
DNA Replication, Ethylene Oxide, Alkylating Agents, Guanine, DNA Repair, Stereochemistry, [SDV]Life Sciences [q-bio], Biology, medicine.disease_cause, Biochemistry, Adduct, 03 medical and health sciences, chemistry.chemical_compound, DNA Adducts, In vivo, [CHIM] Chemical Sciences, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Escherichia coli, [CHIM]Chemical Sciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Propylene oxide, Mutation frequency, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Ethylene oxide, 030302 biochemistry & molecular biology, Cell Biology, 3. Good health, [SDV] Life Sciences [q-bio], chemistry, Epoxy Compounds, Transformation efficiency
Abstract

Adducts formed at the nucleophilic N7 position of guanine are the most abundant lesions produced by alkylating agents such as ethylene oxide (EO) and propylene oxide (PO). In order to investigate the intrinsic mutagenic potential of N 7-alkylguanine adducts, we prepared single-stranded DNA probes containing a single well-defined N 7-alkylguanine adduct under conditions that minimize the presence of depurinated molecules. Following introduction of these probes into Escherichia coli cells, the effect of the N 7-alkylguanine adducts on the efficiency and fidelity of replication was determined. To investigate the effect on replication we monitored the relative transformation efficiency of the lesion containing constructs with respect to the control construct. The methyl adduct was found not to be toxic, while the N 7-(2-hydroxyethyl)guanine ( N 7-heG) and N 7-(2-hydroxypropyl)guanine ( N 7-hpG) adducts reduce the transformation efficiency to ≈70% and 40%, respectively. Within the detection limits of our assay, replication across the N 7-alkylguanine adducts in vivo is essentially error-free, as no mutant colony was observed among ≈300 individual sequenced colonies ( i.e. , mutation frequency

Published
2014

50. Zinc finger oxidation of Fpg/Nei DNA glycosylases by 2-thioxanthine: biochemical and X-ray structural characterization

Author

Alicja Winczura, Didier Gasparutto, Artur Biela, Thomas Carell, Zygmunt Kazimierczuk, Barbara Tudek, Jarosław M. Cieśla, Franck Coste, Martine Guérin, Karola L. Gasteiger, Stéphane Goffinont, Bertrand Castaing, Françoise Culard, Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Ludwig-Maximilians-Universität München, Department of Chemistry and Biochemistry, Institute of Biochemistry and Biophysics PAS, Ul. Pawinskiego 5A, 02-106 Warsaw, Institute of Biochemistry and Biophysics, Warsaw University of Life Sciences (SGGW), Laboratoire Lésions des Acides Nucléiques (LAN), Service de Chimie Inorganique et Biologique (SCIB - UMR E3), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS), Institute of Genetics and Biotechnology, University of Warsaw (UW), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Institute of Biochemistry and Biophysics [Warsaw] (IBB)

Subjects
Models, Molecular, endocrine system diseases, [SDV]Life Sciences [q-bio], Biology, Crystallography, X-Ray, DNA-formamidopyrimidine glycosylase, DNA Glycosylases, chemistry.chemical_compound, Structural Biology, Genetics, Enzyme Inhibitors, chemistry.chemical_classification, Zinc finger, Zinc Fingers, DNA-binding domain, Base excision repair, DNA, Molecular biology, 3. Good health, Zinc, Enzyme, chemistry, Biochemistry, DNA-Formamidopyrimidine Glycosylase, DNA glycosylase, Thioxanthenes, Uncompetitive inhibitor, Oxidation-Reduction
Abstract

International audience; DNA glycosylases from the Fpg/Nei structural superfamily are base excision repair enzymes involved in the removal of a wide variety of mutagen and potentially lethal oxidized purines and pyrimidines. Although involved in genome stability, the recent discovery of synthetic lethal relationships between DNA glycosylases and other pathways highlights the potential of DNA glycosylase inhibitors for future medicinal chemistry development in cancer therapy. By combining biochemical and structural approaches, the physical target of 2-thioxanthine (2TX), an uncompetitive inhibitor of Fpg, was identified. 2TX interacts with the zinc finger (ZnF) DNA binding domain of the enzyme. This explains why the zincless hNEIL1 enzyme is resistant to 2TX. Crystal structures of the enzyme bound to DNA in the presence of 2TX demonstrate that the inhibitor chemically reacts with cysteine thiolates of ZnF and induces the loss of zinc. The molecular mechanism by which 2TX inhibits Fpg may be generalized to all prokaryote and eukaryote ZnF-containing Fpg/Nei-DNA glycosylases. Cell experiments show that 2TX can operate in cellulo on the human Fpg/Nei DNA glycosylases. The atomic elucidation of the determinants for the interaction of 2TX to Fpg provides the foundation for the future design and synthesis of new inhibitors with high efficiency and selectivity.

Published
2014

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