Search

Your search keyword '"Fabrice Confalonieri"' showing total 18 results
Start Over Author "Fabrice Confalonieri" Publication Year Range Last 10 years
18 results on '"Fabrice Confalonieri"'

1. Natural Transformation in Deinococcus radiodurans: A Genetic Analysis Reveals the Major Roles of DprA, DdrB, RecA, RecF, and RecO Proteins

Author

Solenne Ithurbide, Geneviève Coste, Johnny Lisboa, Nicolas Eugénie, Esma Bentchikou, Claire Bouthier de la Tour, Dominique Liger, Fabrice Confalonieri, Suzanne Sommer, Sophie Quevillon-Cheruel, and Pascale Servant

Subjects
Deinococcus radiodurans, natural transformation, DNA uptake, homologous recombination, DprA, RecA, Microbiology, QR1-502
Abstract

Horizontal gene transfer is a major driver of bacterial evolution and adaptation to environmental stresses, occurring notably via transformation of naturally competent organisms. The Deinococcus radiodurans bacterium, characterized by its extreme radioresistance, is also naturally competent. Here, we investigated the role of D. radiodurans players involved in different steps of natural transformation. First, we identified the factors (PilQ, PilD, type IV pilins, PilB, PilT, ComEC-ComEA, and ComF) involved in DNA uptake and DNA translocation across the external and cytoplasmic membranes and showed that the DNA-uptake machinery is similar to that described in the Gram negative bacterium Vibrio cholerae. Then, we studied the involvement of recombination and DNA repair proteins, RecA, RecF, RecO, DprA, and DdrB into the DNA processing steps of D. radiodurans transformation by plasmid and genomic DNA. The transformation frequency of the cells devoid of DprA, a highly conserved protein among competent species, strongly decreased but was not completely abolished whereas it was completely abolished in ΔdprA ΔrecF, ΔdprA ΔrecO, and ΔdprA ΔddrB double mutants. We propose that RecF and RecO, belonging to the recombination mediator complex, and DdrB, a specific deinococcal DNA binding protein, can replace a function played by DprA, or alternatively, act at a different step of recombination with DprA. We also demonstrated that a ΔdprA mutant is as resistant as wild type to various doses of γ-irradiation, suggesting that DprA, and potentially transformation, do not play a major role in D. radiodurans radioresistance.

Published
2020
Full Text
View/download PDF

2. Characterization of the Radiation Desiccation Response Regulon of the Radioresistant Bacterium Deinococcus radiodurans by Integrative Genomic Analyses

Author

Nicolas Eugénie, Yvan Zivanovic, Gaelle Lelandais, Geneviève Coste, Claire Bouthier de la Tour, Esma Bentchikou, Pascale Servant, and Fabrice Confalonieri

Subjects
radioresistance/desiccation, transcriptional regulator, Deinococcus radiodurans, ChIP-seq, RNA-seq, bioinformatic analyses, Cytology, QH573-671
Abstract

Numerous genes are overexpressed in the radioresistant bacterium Deinococcus radiodurans after exposure to radiation or prolonged desiccation. It was shown that the DdrO and IrrE proteins play a major role in regulating the expression of approximately twenty genes. The transcriptional repressor DdrO blocks the expression of these genes under normal growth conditions. After exposure to genotoxic agents, the IrrE metalloprotease cleaves DdrO and relieves gene repression. At present, many questions remain, such as the number of genes regulated by DdrO. Here, we present the first ChIP-seq analysis performed at the genome level in Deinococcus species coupled with RNA-seq, which was achieved in the presence or not of DdrO. We also resequenced our laboratory stock strain of D. radiodurans R1 ATCC 13939 to obtain an accurate reference for read alignments and gene expression quantifications. We highlighted genes that are directly under the control of this transcriptional repressor and showed that the DdrO regulon in D. radiodurans includes numerous other genes than those previously described, including DNA and RNA metabolism proteins. These results thus pave the way to better understand the radioresistance pathways encoded by this bacterium and to compare the stress-induced responses mediated by this pair of proteins in diverse bacteria.

Published
2021
Full Text
View/download PDF

3. Platinum nanoparticles: an exquisite tool to overcome radioresistance

Author

Sha Li, Erika Porcel, Hynd Remita, Sergio Marco, Matthieu Réfrégiers, Murielle Dutertre, Fabrice Confalonieri, and Sandrine Lacombe

Subjects
Metallic nanoparticles, Radioresistance, Radio-enhancement, Radiosensitization, Deinococcus radiodurans, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract

Abstract Backgroud Small metallic nanoparticles are proposed as potential nanodrugs to optimize the performances of radiotherapy. This strategy, based on the enrichment of tumours with nanoparticles to amplify radiation effects in the tumour, aims at increasing the cytopathic effect in tumours while healthy tissue is preserved, an important challenge in radiotherapy. Another major cause of radiotherapy failure is the radioresistance of certain cancers. Surprisingly, the use of nanoparticles to overcome radioresistance has not, to the best of our knowledge, been extensively investigated. The mechanisms of radioresistance have been extensively studied using Deinococcus radiodurans, the most radioresistant organism ever reported, as a model. Methods In this work, we investigated the impact of ultra-small platinum nanoparticles (1.7 nm) on this organism, including uptake, toxicity, and effects on radiation responses. Results We showed that the nanoparticles penetrate D. radiodurans cells, despite the 150 nm cell wall thickness with a minimal inhibition concentration on the order of 4.8 mg L−1. We also found that the nanoparticles amplify gamma ray radiation effects by >40%. Conclusions Finally, this study demonstrates the capacity of metallic nanoparticles to amplify radiation in radioresistant organisms, thus opening the perspective to use nanoparticles not only to improve tumour targeting but also to overcome radioresistance.

Published
2017
Full Text
View/download PDF

4. In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium.

Author

Claire Bouthier de la Tour, Martine Mathieu, Laura Meyer, Pauline Dupaigne, Fanny Passot, Pascale Servant, Suzanne Sommer, Eric Le Cam, and Fabrice Confalonieri

Subjects
Medicine, Science
Abstract

The bacterium Deinococcus radiodurans possesses a set of Deinococcus-specific genes highly induced after DNA damage. Among them, ddrC (dr0003) was recently re-annotated, found to be in the inverse orientation and called A2G07_00380. Here, we report the first in vivo and in vitro characterization of the corrected DdrC protein to better understand its function in irradiated cells. In vivo, the ΔddrC null mutant is sensitive to high doses of UV radiation and the ddrC deletion significantly increases UV-sensitivity of ΔuvrA or ΔuvsE mutant strains. We show that the expression of the DdrC protein is induced after γ-irradiation and is under the control of the regulators, DdrO and IrrE. DdrC is rapidly recruited into the nucleoid of the irradiated cells. In vitro, we show that DdrC is able to bind single- and double-stranded DNA with a preference for the single-stranded DNA but without sequence or shape specificity and protects DNA from various nuclease attacks. DdrC also condenses DNA and promotes circularization of linear DNA. Finally, we show that the purified protein exhibits a DNA strand annealing activity. Altogether, our results suggest that DdrC is a new DNA binding protein with pleiotropic activities. It might maintain the damaged DNA fragments end to end, thus limiting their dispersion and extensive degradation after exposure to ionizing radiation. DdrC might also be an accessory protein that participates in a single strand annealing pathway whose importance in DNA repair becomes apparent when DNA is heavily damaged.

Published
2017
Full Text
View/download PDF

5. Multiple and variable NHEJ-like genes are involved in resistance to DNA damage in Streptomyces ambofaciens

Author

Grégory Hoff, Claire Bertrand, Lingli Zhang, Emilie Piotrowski, Ludovic Chipot, Cyril Bontemps, Fabrice Confalonieri, Stephen McGovern, François Lecointe, Annabelle Thibessard, and Pierre Leblond

Subjects
DNA Damage, DNA Repair, Ligases, Streptomyces, Ku protein, Non homologous end joining, Microbiology, QR1-502
Abstract

Non homologous end-joining (NHEJ) is a double strand break (DSB) repair pathway which does not require any homologous template and can ligate two DNA ends together. The basic bacterial NHEJ machinery involves two partners: the Ku protein, a DNA end binding protein for DSB recognition and the multifunctional LigD protein composed a ligase, a nuclease and a polymerase domain, for end processing and ligation of the broken ends. In silico analyses performed in the 38 sequenced genomes of Streptomyces species revealed the existence of a large panel of NHEJ-like genes. Indeed, ku genes or ligD domain homologues are scattered throughout the genome in multiple copies and can be distinguished in two categories: the core NHEJ gene set constituted of conserved loci and the variable NHEJ gene set constituted of NHEJ-like genes present in only a part of the species. In Streptomyces ambofaciens ATCC 23877, not only the deletion of core genes but also that of variable genes led to an increased sensitivity to DNA damage induced by electron beam irradiation. Multiple mutants of ku, ligase or polymerase encoding genes showed an aggravated phenotype compared to single mutants. Biochemical assays revealed the ability of Ku-like proteins to protect and to stimulate ligation of DNA ends. RT-qPCR and GFP fusion experiments suggested that ku-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination.

Published
2016
Full Text
View/download PDF

6. Identification of new genes contributing to the extreme radioresistance of Deinococcus radiodurans using a Tn5-based transposon mutant library.

Author

Rémi Dulermo, Takefumi Onodera, Geneviève Coste, Fanny Passot, Murielle Dutertre, Martine Porteron, Fabrice Confalonieri, Suzanne Sommer, and Cécile Pasternak

Subjects
Medicine, Science
Abstract

Here, we have developed an extremely efficient in vivo Tn5-based mutagenesis procedure to construct a Deinococcus radiodurans insertion mutant library subsequently screened for sensitivity to genotoxic agents such as γ and UV radiations or mitomycin C. The genes inactivated in radiosensitive mutants belong to various functional categories, including DNA repair functions, stress responses, signal transduction, membrane transport, several metabolic pathways, and genes of unknown function. Interestingly, preliminary characterization of previously undescribed radiosensitive mutants suggests the contribution of cyclic di-AMP signaling in the recovery of D. radiodurans cells from genotoxic stresses, probably by modulating several pathways involved in the overall cell response. Our analyses also point out a new transcriptional regulator belonging to the GntR family, encoded by DR0265, and a predicted RNase belonging to the newly described Y family, both contributing to the extreme radioresistance of D. radiodurans. Altogether, this work has revealed new cell responses involved either directly or indirectly in repair of various cell damage and confirmed that D. radiodurans extreme radiation resistance is determined by a multiplicity of pathways acting as a complex network.

Published
2015
Full Text
View/download PDF

7. Structural and functional determinants of the archaeal 8-oxoguanine-DNA glycosylase AGOG for DNA damage recognition and processing

Author

Franck, Coste, Goffinont, Stéphane, Cros, Julien, Gaudon, Virginie, Martine, Guérin, Garnier, Norbert, Fabrice, Confalonieri, Flament, Didier, Suskiewicz, Marcin Jozef, Bertrand, Castaing, Centre de biophysique moléculaire (CBM), 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), 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), Biologie et Ecologie des Ecosystèmes Marins Profonds (BEEP), and Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM]
Abstract

International audience; Abstract 8-Oxoguanine (GO) is a major purine oxidation product in DNA. Because of its highly mutagenic properties, GO absolutely must be eliminated from DNA. To do this, aerobic and anaerobic organisms from the three kingdoms of life have evolved repair mechanisms to prevent its deleterious effect on genetic integrity. The major way to remove GO is the base excision repair pathway, usually initiated by a GO-DNA glycosylase. First identified in bacteria (Fpg) and eukaryotes (OGG1), GO-DNA glycosylases were more recently identified in archaea (OGG2 and AGOG). AGOG is the less documented enzyme and its mode of damage recognition and removing remains to be clarified at the molecular and atomic levels. This study presents a complete structural characterisation of apo AGOGs from Pyrococcus abyssi (Pab) and Thermococcus gammatolerans (Tga) and the first structure of Pab-AGOG bound to lesion-containing single- or double-stranded DNA. By combining X-ray structure analysis, site directed mutagenesis and biochemistry experiments, we identified key amino acid residues of AGOGs responsible for the specific recognition of the lesion and the base opposite the lesion and for catalysis. Moreover, a unique binding mode of GO, involving double base flipping, never observed for any other DNA glycosylases, is revealed. In addition to unravelling the properties of AGOGs, our study, through comparative biochemical and structural analysis, offers new insights into the evolutionary plasticity of DNA glycosylases across all three kingdoms of life.

Published
2022

8. Structural and functional characterization of DdrC, a novel DNA damage-induced nucleoid associated protein involved in DNA compaction

Author

Anne-Sophie Banneville, Claire Bouthier de la Tour, Cécilia Hognon, Joanna Timmins, Pascale Servant, Antonio Monari, Jean-Marie Teulon, François Dehez, Aline Le Roy, Jacques-Philippe Colletier, Jean-Luc Pellequer, Fabrice Confalonieri, 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), 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 de Physique et Chimie Théoriques (LPCT), Institut de Chimie du CNRS (INC)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Interfaces, Traitements, Organisation et Dynamique des Systèmes (ITODYS (UMR_7086)), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), The ANR (Agence Nationale de la Recherche) and CGI (Commissariat à l’Investissement d’Avenir) are gratefully acknowledged for their financial support of this work through Labex SEAM (Science and Engineering for Advanced Materials and devices): ANR-11-LABX-086 and ANR-11-IDEX-05-02, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), and Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)

Subjects
0303 health sciences, DNA Repair, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, 030306 microbiology, Chemistry, DNA damage, DNA repair, Deinococcus radiodurans, biology.organism_classification, Cell biology, DNA binding site, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Genetics, Nucleoid, A-DNA, Deinococcus, DNA, Circular, Gene, DNA, DNA Damage, 030304 developmental biology
Abstract

Deinococcus radiodurans is a spherical bacterium well-known for its outstanding resistance to DNA-damaging agents. Exposure to such agents leads to drastic changes in the transcriptome of D. radiodurans. In particular, four Deinococcus-specific genes, known as DNA Damage Response genes, are strongly up-regulated and have been shown to contribute to the resistance phenotype of D. radiodurans. One of these, DdrC, is expressed shortly after exposure to γ-radiation and is rapidly recruited to the nucleoid. In vitro, DdrC has been shown to compact circular DNA, circularize linear DNA, anneal complementary DNA strands and protect DNA from nucleases. To shed light on the possible functions of DdrC in D. radiodurans, we determined the crystal structure of the domain-swapped DdrC dimer at a resolution of 2.2 Å and further characterized its DNA binding and compaction properties. Notably, we show that DdrC bears two asymmetric DNA binding sites located on either side of the dimer and can modulate the topology and level of compaction of circular DNA. These findings suggest that DdrC may be a DNA damage-induced nucleoid-associated protein that enhances nucleoid compaction to limit the dispersion of the fragmented genome and facilitate DNA repair after exposure to severe DNA damaging conditions.

Published
2021

9. Characterization of the Radiation Desiccation Response Regulon of the Radioresistant Bacterium Deinococcus radiodurans by Integrative Genomic Analyses

Author

Gaëlle Lelandais, C. Bouthier de la Tour, Esma Bentchikou, Nicolas Eugénie, Yvan Zivanovic, Geneviève Coste, Pascale Servant, Fabrice Confalonieri, 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), and ANR-19-CE12-0010,NOVOREP,Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress(2019)

Subjects
QH301-705.5, RNA-Seq, Biology, Regulon, Genome, Article, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Deinococcus radiodurans, Radioresistance, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Gene expression, Homologous chromosome, Transcriptional regulation, Deinococcus, Biology (General), Gene, 030304 developmental biology, transcriptional regulator, Genetics, Metalloproteinase, 0303 health sciences, bioinformatic analyses, 030306 microbiology, Gene Expression Regulation, Bacterial, Genomics, General Medicine, biology.organism_classification, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, ChIP-seq, chemistry, RNA-seq, radioresistance/desiccation, Bacteria, DNA, DNA Damage, Transcription Factors
Abstract

Numerous genes are overexpressed in the radioresistant bacterium Deinococcus radiodurans after exposure to radiation or prolonged desiccation. The DdrO and IrrE proteins play a major role in regulating the expression of approximately predicted twenty of these genes. The transcriptional repressor DdrO blocks the expression of these genes under normal growth conditions. After exposure to genotoxic agents, the IrrE metalloprotease cleaves DdrO and relieves gene repression. Bioinformatic analyzes showed that this mechanism seems to be conserved in several species of Deinococcus, but many questions remain as such the number of genes regulated by DdrO. Here, by RNA-seq and CHiP-seq assays performed at a genome-wide scale coupled with bioinformatic analyses, we show that, the DdrO regulon in D. radiodurans includes many other genes than those previously described. These results thus pave the way to better understand the radioresistance mechanisms encoded by this bacterium.Author SummaryThe main response pathway to genotoxic conditions in the radioresistant bacterium Deinococcus radiodurans is regulated by the constitutively expressed metalloprotease IrrE that cleaves the transcriptional repressor DdrO, leading to the expression of the genes repressed by DdrO. One of the major goals to better understand how pathways involved in radioresistance are coordinated into this fascinating bacterium is to highlight genes regulated by DdrO. In this study, we mapped in vivo the DdrO regulon in D. radiodurans by using two genome-scale approaches, ChIP-seq and RNA-seq analyses, coupled with bioinformatic analyses. As homologs of these two proteins are also found in many other bacteria, these results also pave the way to compare the stress-induced responses mediated by this couple of proteins in diverse bacteria.

Published
2021

10. Characterization of the DdrD protein from the extremely radioresistant bacterium Deinococcus radiodurans

Author

Claire Bouthier de la Tour, Pascale Servant, Cédric Norais, Geneviève Coste, Fabrice Confalonieri, Martine Mathieu, 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), and University of Wisconsin-Madison

Subjects
Protein family, DNA Repair, DNA damage, Mutant, DdrD, DNA damage response, Microbiology, DNA-binding protein, 03 medical and health sciences, chemistry.chemical_compound, Plasmid, Bacterial Proteins, Deinococcus radiodurans, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], 030304 developmental biology, 0303 health sciences, Original Paper, biology, 030306 microbiology, General Medicine, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, DNA binding protein, Cell biology, Regulon, chemistry, Molecular Medicine, Deinococcus, DNA, DNA Damage, Plasmids
Abstract

Here, we report the in vitro and in vivo characterization of the DdrD protein from the extraordinary stress-resistant bacterium, D. radiodurans. DdrD is one of the most highly induced proteins following cellular irradiation or desiccation. We confirm that DdrD belongs to the Radiation Desiccation Response (RDR) regulon protein family whose expression is regulated by the IrrE/DdrO proteins after DNA damage. We show that DdrD is a DNA binding protein that binds to single-stranded DNA In vitro, but not to duplex DNA unless it has a 5′ single-stranded extension. In vivo, we observed no significant effect of the absence of DdrD on the survival of D. radiodurans cells after exposure to γ-rays or UV irradiation in different genetic contexts. However, genome reassembly is affected in a ∆ddrD mutant when cells recover from irradiation in the absence of nutrients. Thus, DdrD likely contributes to genome reconstitution after irradiation, but only under starvation conditions. Lastly, we show that the absence of the DdrD protein partially restores the frequency of plasmid transformation of a ∆ddrB mutant, suggesting that DdrD could also be involved in biological processes other than the response to DNA damage.

Published
2021

11. Natural Transformation in Deinococcus radiodurans: A Genetic Analysis Reveals the Major Roles of DprA, DdrB, RecA, RecF, and RecO Proteins

Author

Dominique Liger, Solenne Ithurbide, Fabrice Confalonieri, Suzanne Sommer, Sophie Quevillon-Cheruel, Esma Bentchikou, Pascale Servant, Nicolas Eugénie, Johnny Lisboa, Claire Bouthier de la Tour, Geneviève Coste, 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), Radiorésistance des bactéries et des archées (RBA), Département Biologie des Génomes (DBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-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)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Fonction et Architecture des Assemblages Macromoléculaires (FAAM), Département Biochimie, Biophysique et Biologie Structurale (B3S), and Instituto de Investigação e Inovação em Saúde

Subjects
Microbiology (medical), RecFOR, DNA repair, [SDV]Life Sciences [q-bio], Mutant, lcsh:QR1-502, homologous recombination, Biology, DdrB, Microbiology, natural transformation, lcsh:Microbiology, 03 medical and health sciences, chemistry.chemical_compound, Plasmid, Deinococcus radiodurans, 0502 Environmental Science and Management, 0503 Soil Sciences, 0605 Microbiology, 030304 developmental biology, Original Research, Genetics, 0303 health sciences, RecA, 030306 microbiology, DNA uptake, Wild type, biology.organism_classification, Transformation (genetics), chemistry, DprA, Homologous recombination, DNA
Abstract

Horizontal gene transfer is a major driver of bacterial evolution and adaptation to environmental stresses, occurring notably via transformation of naturally competent organisms. The Deinococcus radiodurans bacterium, characterized by its extreme radioresistance, is also naturally competent. Here, we investigated the role of D. radiodurans players involved in different steps of natural transformation. First, we identified the factors (PilQ, PilD, type IV pilins, PilB, PilT, ComEC-ComEA, and ComF) involved in DNA uptake and DNA translocation across the external and cytoplasmic membranes and showed that the DNA-uptake machinery is similar to that described in the Gram negative bacterium Vibrio cholerae. Then, we studied the involvement of recombination and DNA repair proteins, RecA, RecF, RecO, DprA, and DdrB into the DNA processing steps of D. radiodurans transformation by plasmid and genomic DNA. The transformation frequency of the cells devoid of DprA, a highly conserved protein among competent species, strongly decreased but was not completely abolished whereas it was completely abolished in ΔdprA ΔrecF, ΔdprA ΔrecO, and ΔdprA ΔddrB double mutants. We propose that RecF and RecO, belonging to the recombination mediator complex, and DdrB, a specific deinococcal DNA binding protein, can replace a function played by DprA, or alternatively, act at a different step of recombination with DprA. We also demonstrated that a ΔdprA mutant is as resistant as wild type to various doses of γ-irradiation, suggesting that DprA, and potentially transformation, do not play a major role in D. radiodurans radioresistance. SI, NE, and JL were supported by a Ph.D. fellowship from the French Ministry of Education. This work was supported by the Centre National de la Recherche Scientifique, the University Paris-Sud, the Agence Nationale de la Recherche (ANR Radioresistance-11-BSV3-01701 to SS), and by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01.

Published
2020

12. Crystal structure of the transcriptional repressor DdrO: insight into the metalloprotease/repressor-controlled radiation response in Deinococcus

Author

Philippe Roche, Fabrice Confalonieri, Pascale Servant, Romaric Magerand, David Lemaire, David Pignol, François Parcy, Pascal Arnoux, Géraldine Brandelet, Raquel Martin-Arevalillo, Laurence Blanchard, Jade Doloy, Nicolas Eugénie, Arjan de Groot, Marina I. Siponen, Renaud Dumas, Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-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)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-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 Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Radiorésistance des bactéries et des archées (RBA), Département Biologie des Génomes (DBG), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Régulateurs du développement de la fleur (Flo_RE), Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-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 National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Interactions Protéine Métal (IPM), Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC), ANR-19-CE12-0010,NOVOREP,Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-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)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-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 National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-19-CE12-0010,NOVOREP,Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress(2019), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-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])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Université Paris-Saclay, Université Grenoble Alpes (UGA)-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 National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-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 National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), MARAVAL, Alexandra, and Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress - - NOVOREP2019 - ANR-19-CE12-0010 - AAPG2019 - VALID

Subjects
Models, Molecular, DNA damage, [SDV]Life Sciences [q-bio], Repressor, [SDV.CAN]Life Sciences [q-bio]/Cancer, Cleavage (embryo), Crystallography, X-Ray, DNA-binding protein, Protein Structure, Secondary, 03 medical and health sciences, Protein structure, Structural Biology, Stress, Physiological, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Genetics, Deinococcus, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Peptide sequence, 030304 developmental biology, 0303 health sciences, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, biology, 030306 microbiology, Gene Expression Regulation, Bacterial, biology.organism_classification, Protein Structure, Tertiary, [SDV] Life Sciences [q-bio], Repressor Proteins, Biophysics, Metalloproteases, Alpha helix, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, DNA Damage, Transcription Factors
Abstract

Exposure to harmful conditions such as radiation and desiccation induce oxidative stress and DNA damage. In radiation-resistant Deinococcus bacteria, the radiation/desiccation response is controlled by two proteins: the XRE family transcriptional repressor DdrO and the COG2856 metalloprotease IrrE. The latter cleaves and inactivates DdrO. Here, we report the biochemical characterization and crystal structure of DdrO, which is the first structure of a XRE protein targeted by a COG2856 protein. DdrO is composed of two domains that fold independently and are separated by a flexible linker. The N-terminal domain corresponds to the DNA-binding domain. The C-terminal domain, containing three alpha helices arranged in a novel fold, is required for DdrO dimerization. Cleavage by IrrE occurs in the loop between the last two helices of DdrO and abolishes dimerization and DNA binding. The cleavage site is hidden in the DdrO dimer structure, indicating that IrrE cleaves DdrO monomers or that the interaction with IrrE induces a structural change rendering accessible the cleavage site. Predicted COG2856/XRE regulatory protein pairs are found in many bacteria, and available data suggest two different molecular mechanisms for stress-induced gene expression: COG2856 protein-mediated cleavage or inhibition of oligomerization without cleavage of the XRE repressor.

Published
2019

13. DdrI, a cAMP Receptor Protein Family Member, Acts as a Major Regulator for Adaptation of Deinococcus radiodurans to Various Stresses

Author

Jacques Oberto, Laura Meyer, Suzanne Sommer, Geneviève Coste, Fabrice Confalonieri, Cécile Pasternak, Pascale Servant, Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Biologie Cellulaire des Archées (ARCHEE), Département Microbiologie (Dpt Microbio), 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)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-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)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Cyclic AMP Receptor Protein, DNA repair, Ultraviolet Rays, 030106 microbiology, Population, Microbiology, Plasmid maintenance, 03 medical and health sciences, Bacterial Proteins, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Heat shock, education, Molecular Biology, Gene, Transcription factor, ComputingMilieux_MISCELLANEOUS, education.field_of_study, biology, Deinococcus radiodurans, Gene Expression Regulation, Bacterial, biology.organism_classification, Adaptation, Physiological, Cell biology, cAMP receptor protein, biology.protein, Deinococcus, Research Article
Abstract

The D NA d amage r esponse ddrI gene encodes a transcription regulator belonging to the cAMP receptor protein (CRP) family. Cells devoid of the DdrI protein exhibit a pleiotropic phenotype, including growth defects and sensitivity to DNA-damaging agents and to oxidative stress. Here, we show that the absence of the DdrI protein also confers sensitivity to heat shock treatment, and several genes involved in heat shock response were shown to be upregulated in a DdrI-dependent manner. Interestingly, expression of the Escherichia coli CRP partially compensates for the absence of the DdrI protein. Microscopic observations of Δ ddrI mutant cells revealed an increased proportion of two-tetrad and anucleated cells in the population compared to the wild-type strain, indicating that DdrI is crucial for the completion of cell division and/or chromosome segregation. We show that DdrI is also involved in the megaplasmid MP1 stability and in efficient plasmid transformation by facilitating the maintenance of the incoming plasmid in the cell. The in silico prediction of putative DdrI binding sites in the D. radiodurans genome suggests that hundreds of genes, belonging to several functional groups, may be regulated by DdrI. In addition, the DdrI protein absolutely requires cAMP for in vitro binding to specific target sequences, and it acts as a dimer. All these data underline the major role of DdrI in D. radiodurans physiology under normal and stress conditions by regulating, both directly and indirectly, a cohort of genes involved in various cellular processes, including central metabolism and specific responses to diverse harmful environments. IMPORTANCE Deinococcus radiodurans has been extensively studied to elucidate the molecular mechanisms responsible for its exceptional ability to withstand lethal effects of various DNA-damaging agents. A complex network, including efficient DNA repair, protein protection against oxidation, and diverse metabolic pathways, plays a crucial role for its radioresistance. The regulatory networks orchestrating these various pathways are still missing. Our data provide new insights into the crucial contribution of the transcription factor DdrI for the D. radiodurans ability to withstand harmful conditions, including UV radiation, mitomycin C treatment, heat shock, and oxidative stress. Finally, we highlight that DdrI is also required for accurate cell division, for maintenance of plasmid replicons, and for central metabolism processes responsible for the overall cell physiology.

Published
2018

14. In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

Author

Pauline Dupaigne, Pascale Servant, Fabrice Confalonieri, Martine Mathieu, Eric Le Cam, Claire Bouthier de la Tour, Fanny Passot, Suzanne Sommer, Laura Meyer, 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), Radiorésistance des bactéries et des archées (RBA), Département Biologie des Génomes (DBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-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)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-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), Institut Gustave Roussy (IGR), Interactions moléculaires et cancer (IMC (UMR 8126)), and Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, DNA Repair, Hydrolases, [SDV]Life Sciences [q-bio], Oligonucleotides, lcsh:Medicine, Biochemistry, DNA annealing, chemistry.chemical_compound, Annealing activity, lcsh:Science, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, biology, Nucleotides, Physics, Recombinant Proteins, Circular DNA, Enzymes, Nucleic acids, Physical sciences, Nucleic acid thermodynamics, Deinococcus, Research Article, Plasmids, Forms of DNA, Nucleases, Ultraviolet Rays, DNA damage, DNA repair, Annealing (genetics), Biophysics, DNA, Single-Stranded, 03 medical and health sciences, Bacterial Proteins, Single-Strand Annealing, DNA-binding proteins, Genetics, Nucleoid, Protein Structure, Quaternary, Biology and life sciences, Oligonucleotide, lcsh:R, Proteins, Dose-Response Relationship, Radiation, Deinococcus radiodurans, DNA, biology.organism_classification, Molecular biology, 030104 developmental biology, chemistry, Gamma Rays, Enzymology, lcsh:Q, Protein Multimerization, Gene Deletion, In vitro recombination
Abstract

International audience; The bacterium Deinococcus radiodurans possesses a set of Deinococcus-specific genes highly induced after DNA damage. Among them, ddrC (dr0003) was recently re-annotated, found to be in the inverse orientation and called A2G07\₀0380. Here, we report the first in vivo and in vitro characterization of the corrected DdrC protein to better understand its function in irradiated cells. In vivo, the ΔddrC null mutant is sensitive to high doses of UV radiation and the ddrC deletion significantly increases UV-sensitivity of ΔuvrA or ΔuvsE mutant strains. We show that the expression of the DdrC protein is induced after γ-irradiation and is under the control of the regulators, DdrO and IrrE. DdrC is rapidly recruited into the nucleoid of the irradiated cells. In vitro, we show that DdrC is able to bind single- and double-stranded DNA with a preference for the single-stranded DNA but without sequence or shape specificity and protects DNA from various nuclease attacks. DdrC also condenses DNA and promotes circularization of linear DNA. Finally, we show that the purified protein exhibits a DNA strand annealing activity. Altogether, our results suggest that DdrC is a new DNA binding protein with pleiotropic activities. It might maintain the damaged DNA fragments end to end, thus limiting their dispersion and extensive degradation after exposure to ionizing radiation. DdrC might also be an accessory protein that participates in a single strand annealing pathway whose importance in DNA repair becomes apparent when DNA is heavily damaged.

Published
2017

15. 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

16. Prioritizing targets for structural biology through the lens of proteomics: The archaeal protein TGAM_1934 from Thermococcus gammatolerans

Author

Valérie Aloin, Fabrice Confalonieri, Alain Dedieu, Karine de Guillen, Bernard Fernandez, Jean-Baptiste Boyer, Arnaud Lagorce, Jean Armengaud, Christian Roumestand, Yinshan Yang, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Biochimie des Systèmes Perturbés (LBSP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Radiorésistance des bactéries et des archées (RBA), Département Biologie des Génomes (DBG), 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)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-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)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] ( CBS ), Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de Biochimie des Systèmes Perturbés ( LBSP ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Institut de génétique et microbiologie [Orsay] ( IGM ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre National de la Recherche Scientifique ( CNRS ), Radiorésistance des bactéries et des archées ( RBA ), Département Biologie des Génomes ( DBG ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Proteomics, Protein Conformation, Archaeal Proteins, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Quantitative proteomics, High-throughput proteomics, Computational biology, Biochemistry, Microbiology, Protein structure, Iron-Binding Proteins, Amino Acid Sequence, Nuclear Magnetic Resonance, Biomolecular, Molecular Biology, Peptide sequence, biology, [ SDV ] Life Sciences [q-bio], Genome project, computer.file_format, ORFan, biology.organism_classification, Protein Data Bank, Molecular biology, Thermococcus, Structural biology, Protein evolution, NMR structure, computer, Genome annotation
Abstract

International audience; ORFans are hypothetical proteins lacking any significant sequence similarity with other proteins. Here, we highlighted by quantitative proteomics the TGAM_1934 ORFan from the hyperradioresistant Thermococcus gammatolerans archaeon as one of the most abundant hypothetical proteins. This protein has been selected as a priority target for structure determination on the basis of its abundance in three cellular conditions. Its solution structure has been determined using multidimensional heteronuclear NMR spectroscopy. TGAM_1934 displays an original fold, although sharing some similarities with the 3D structure of the bacterial ortholog of frataxin, CyaY, a protein conserved in bacteria and eukaryotes and involved in iron-sulfur cluster biogenesis. These results highlight the potential of structural proteomics in prioritizing ORFan targets for structure determination based on quantitative proteomics data. The proteomic data and structure coordinates have been deposited to the ProteomeXchange with identifier PXD000402 (http://proteomecentral.proteomexchange.org/dataset/PXD000402) and Protein Data Bank under the accession number 2mcf, respectively.

Published
2015

17. Etude de machines moléculaires impliquées dans la dynamique des génomes bactériens et phagiques

Author

Lecointe, François, MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université Paris Saclay, and Fabrice Confalonieri

Subjects
Single strand annealing, Molecular machines, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Appariement simple brin, Double strand break repair, Machines moléculaires, Bacterial and bacteriophage evolution, Evolution des bactéries et des bactériophages, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, NHEJ, réparation des cassures double brin
Abstract

Les différentes recherches que je mène depuis ma thèse portent sur la caractérisation de mécanismes mis en jeu dans différents aspects du métabolisme des acides nucléiques chez les microbes. L'étude des protéines isolées impliquées dans ces processus, bien qu'essentielle, s'est avérée rapidement limitée pour comprendre les mécanismes dans leur ensemble et expliquer des processus, même simples. Suite à mon recrutement à l'INRA, je me suis donc focalisé sur l'identification des machines moléculaires, i.e. des complexes protéiques ou nucléoprotéiques, assurant ces mécanismes, et sur la caractérisation des interactions assurant leur cohésion et leur dynamique.J’ai tout d’abord étudié la recombinaison homologue et le redémarrage de la réplication chez Bacillus subtilis via l'étude du partenariat de la protéine de fixation à l'ADN simple brin, SSB, ainsi que la voie de réparation par recombinaison non homologue (NHEJ), via l'étude de la protéine Ku et de son partenaire LigD. J’ai alors pu établir un modèle de leurs rôles respectifs dans ces mécanismes de réparation.Plus récemment, je me suis intéressé aux machines moléculaires impliquées dans les mécanismes de recombinaison de l’ADN chez les phages (virus de bactérie). En effet, ces entités suscitent un regain d’intérêt ces dernières années en raison de leur potentiel thérapeutique pour lutter contre le phénomène d'antibiorésistance des bactéries pathogènes, apparu rapidement après l'utilisation massive des antibiotiques au cours du XXème siècle, et qui représente désormais un problème de santé publique majeur. Un inconvénient possible à l'emploi de la phagothérapie est lié à l’évolution génétique rapide des phages, due en partie à leur capacité à coder des systèmes de recombinaison de l'ADN particulièrement divers et efficaces. Ces systèmes restent cependant pour la plupart à caractériser. De plus, et contrairement aux organismes cellulaires, ces systèmes ne sont pas ubiquitaires, soulevant la question de leur rôle, à la fois pour le phage lui-même, mais également potentiellement pour son hôte bactérien.Ainsi, ma recherche actuelle vise premièrement à caractériser des mécanismes de recombinaison phagiques originaux, via l'étude des machines impliquées. Deuxièmement, je cherche à comprendre l'impact de ces systèmes de réparation sur le cycle de vie des phages et en quoi ils influencent la physiologie des bactéries à l'état lysogénique.Pour répondre à ces objectifs, deux machines moléculaires sont en cours d'étude. La première implique la recombinase Sak4 et sa SSB, codées par le phage HK620 d'Escherichia coli. Nous avons montré que l'association des deux est requise pour une recombinaison homologue par appariement simple brin efficace. Cette caractéristique est inédite puisque les SSB sont connues pour inhiber cette activité chez les autres recombinases de la même famille (type RAD51), faisant de Sak4 une recombinase particulièrement intéressante à caractériser d'un point de vue évolution et fonction. Nous cherchons aussi à déterminer l'influence de ce couple dans le processus d'induction du cycle lytique du phage. La deuxième machine est composée a minima des protéines Mu-Gam et RecT du phage Gally, potentiellement impliquées respectivement dans des évènements de recombinaison non homologue et homologue. Cette combinatoire au sein d'un même phage et la résultante en terme de mécanisme de recombinaison n'a jamais été étudiée chez les phages et nos premiers résultats indiquent que ces deux protéines fonctionnent ensemble dans un mécanisme en cours de caractérisation. Ce mécanisme n'influe que très peu sur la capacité de Gally à produire des virions. A l'inverse, des données préliminaires nous indique que Mu-Gam et RecT pourraient être impliquées dans la virulence de la souche E. coli LF82, une souche porteuse du prophage Gally et qui est impliquée dans la maladie de Crohn. Nous cherchons maintenant à valider ces résultats et à comprendre le lien entre recombinaison phagique et virulence bactérienne.L'ensemble des résultats générés au cours de ces études permet de mieux comprendre les mécanismes de réparation de l'ADN chez les phages, et une partie de ces connaissances devraient être transposables chez les organismes cellulaires. Ces études impliquent également que des mécanismes de réparation phagiques, à l'origine de leur évolution et du bon déroulement de leur cycle de vie, pourraient être adoptés par leur hôte bactérien pour leur propre bénéfice dans des environnements génotoxiques.

Published
2021

18. Structural and functional determinants of the archaeal 8-oxoguanine-DNA glycosylase AGOG for DNA damage recognition and processing.

Author

Franck C, Stéphane G, Julien C, Virginie G, Martine G, Norbert G, Fabrice C, Didier F, Josef SM, and Bertrand C

Subjects
DNA Damage, DNA Repair, DNA genetics, DNA-Formamidopyrimidine Glycosylase genetics, DNA-Formamidopyrimidine Glycosylase metabolism, DNA Glycosylases metabolism, Thermococcus genetics
Abstract

8-Oxoguanine (GO) is a major purine oxidation product in DNA. Because of its highly mutagenic properties, GO absolutely must be eliminated from DNA. To do this, aerobic and anaerobic organisms from the three kingdoms of life have evolved repair mechanisms to prevent its deleterious effect on genetic integrity. The major way to remove GO is the base excision repair pathway, usually initiated by a GO-DNA glycosylase. First identified in bacteria (Fpg) and eukaryotes (OGG1), GO-DNA glycosylases were more recently identified in archaea (OGG2 and AGOG). AGOG is the less documented enzyme and its mode of damage recognition and removing remains to be clarified at the molecular and atomic levels. This study presents a complete structural characterisation of apo AGOGs from Pyrococcus abyssi (Pab) and Thermococcus gammatolerans (Tga) and the first structure of Pab-AGOG bound to lesion-containing single- or double-stranded DNA. By combining X-ray structure analysis, site directed mutagenesis and biochemistry experiments, we identified key amino acid residues of AGOGs responsible for the specific recognition of the lesion and the base opposite the lesion and for catalysis. Moreover, a unique binding mode of GO, involving double base flipping, never observed for any other DNA glycosylases, is revealed. In addition to unravelling the properties of AGOGs, our study, through comparative biochemical and structural analysis, offers new insights into the evolutionary plasticity of DNA glycosylases across all three kingdoms of life., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Published
2022
Full Text
View/download PDF

Catalog

Books, media, physical & digital resources
See catalog results