44 results on '"Joshua A. Sommers"'
Search Results
2. Biochemical analysis of SARS-CoV-2 Nsp13 helicase implicated in COVID-19 and factors that regulate its catalytic functions
- Author
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Joshua A. Sommers, Lorin N. Loftus, Martin P. Jones, Rebecca A. Lee, Caitlin E. Haren, Adaira J. Dumm, and Robert M. Brosh
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Cell Biology ,Molecular Biology ,Biochemistry - Published
- 2023
3. Mitochondrial genetic variation is enriched in G-quadruplex regions that stall DNA synthesis in vitro
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Luigi Ferrucci, Katrina N Estep, Thomas J. Butler, Robert W. Maul, David Schlessinger, Andrew R. Wood, Marcus A. Tuke, Joshua A. Sommers, Thomas A. Guilliam, Francesco Cucca, Daniel F. Bogenhagen, Alicia K. Byrd, Aidan J. Doherty, Stefania Bandinelli, Robert M. Brosh, Sanjay Kumar Bharti, Elena Yakubovskaya, Miguel Garcia-Diaz, Ann Zenobia Moore, Jun Ding, and Kevin D. Raney
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DNA Replication ,Mitochondrial DNA ,Guanine ,DNA Primase ,DNA-Directed DNA Polymerase ,DNA, Mitochondrial ,Genome ,DNA sequencing ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Genetics ,Humans ,Molecular Biology ,Genetics (clinical) ,030304 developmental biology ,0303 health sciences ,Whole Genome Sequencing ,biology ,DNA synthesis ,DNA Helicases ,DNA replication ,Helicase ,General Medicine ,Multifunctional Enzymes ,DNA Polymerase gamma ,Mitochondria ,Cell biology ,G-Quadruplexes ,Italy ,chemistry ,Mutagenesis ,Genome, Mitochondrial ,Mutation ,biology.protein ,Nucleic Acid Conformation ,General Article ,030217 neurology & neurosurgery ,DNA ,Mitochondrial DNA replication - Abstract
As the powerhouses of the eukaryotic cell, mitochondria must maintain their genomes which encode proteins essential for energy production. Mitochondria are characterized by guanine-rich DNA sequences that spontaneously form unusual three-dimensional structures known as G-quadruplexes (G4). G4 structures can be problematic for the essential processes of DNA replication and transcription because they deter normal progression of the enzymatic-driven processes. In this study, we addressed the hypothesis that mitochondrial G4 is a source of mutagenesis leading to base-pair substitutions. Our computational analysis of 2757 individual genomes from two Italian population cohorts (SardiNIA and InCHIANTI) revealed a statistically significant enrichment of mitochondrial mutations within sequences corresponding to stable G4 DNA structures. Guided by the computational analysis results, we designed biochemical reconstitution experiments and demonstrated that DNA synthesis by two known mitochondrial DNA polymerases (Pol γ, PrimPol) in vitro was strongly blocked by representative stable G4 mitochondrial DNA structures, which could be overcome in a specific manner by the ATP-dependent G4-resolving helicase Pif1. However, error-prone DNA synthesis by PrimPol using the G4 template sequence persisted even in the presence of Pif1. Altogether, our results suggest that genetic variation is enriched in G-quadruplex regions that impede mitochondrial DNA replication.
- Published
- 2020
4. DNA polymerase β outperforms DNA polymerase γ in key mitochondrial base excision repair activities
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Stephanie L. Baringer, Deborah L. Croteau, Beverly A. Baptiste, Tomasz Kulikowicz, Robert M. Brosh, Vilhelm A. Bohr, and Joshua A. Sommers
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DNA Repair ,DNA polymerase ,DNA polymerase beta ,Mitochondrion ,Biochemistry ,DNA, Mitochondrial ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Animals ,Molecular Biology ,Polymerase ,DNA Polymerase beta ,030304 developmental biology ,0303 health sciences ,biology ,Helicase ,Cell Biology ,Base excision repair ,Nuclear DNA ,Cell biology ,DNA Polymerase gamma ,Mitochondria ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,DNA ,DNA Damage - Abstract
DNA polymerase beta (POLβ), well known for its role in nuclear DNA base excision repair (BER), has been shown to be present in the mitochondria of several different cell types. Here we present a side-by-side comparison of BER activities of POLβ and POLγ, the mitochondrial replicative polymerase, previously thought to be the only mitochondrial polymerase. We find that POLβ is significantly more proficient at single-nucleotide gap filling, both in substrates with ends that require polymerase processing, and those that do not. We also show that POLβ has a helicase-independent functional interaction with the mitochondrial helicase, TWINKLE. This interaction stimulates strand-displacement synthesis, but not single-nucleotide gap filling. Importantly, we find that purified mitochondrial extracts from cells lacking POLβ are severely deficient in processing BER intermediates, suggesting that mitochondrially localized DNA POLβ may be critical for cells with high energetic demands that produce greater levels of oxidative stress and therefore depend upon efficient BER for mitochondrial health.
- Published
- 2020
5. Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase
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Sean M. Carney, Joshua A. Sommers, Elena Yakubovskaya, Sanjay Kumar Bharti, Robert M. Brosh, Jack D. Crouch, Michael A. Trakselis, Irfan Khan, and Miguel Garcia-Diaz
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0301 basic medicine ,Mitochondrial DNA ,030102 biochemistry & molecular biology ,biology ,DNA repair ,DNA damage ,DNA replication ,Helicase ,Cell Biology ,Biochemistry ,Molecular biology ,Branch migration ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,biology.protein ,Replisome ,Molecular Biology ,DNA - Abstract
Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5′ to 3′ helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5′ or 3′ single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5′ to 3′ directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding.
- Published
- 2016
6. Catalytic Strand Separation by RECQ1 Is Required for RPA-Mediated Response to Replication Stress
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Michael M. Seidman, Taraswi Banerjee, Robert M. Brosh, Joshua A. Sommers, and Jing Huang
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DNA Replication ,Genome instability ,DNA Repair ,DNA repair ,DNA damage ,RecQ helicase ,Genomic Instability ,Article ,General Biochemistry, Genetics and Molecular Biology ,Substrate Specificity ,Stress, Physiological ,Cell Line, Tumor ,Replication Protein A ,Humans ,Replication protein A ,Cells, Cultured ,RecQ Helicases ,Agricultural and Biological Sciences(all) ,biology ,Biochemistry, Genetics and Molecular Biology(all) ,DNA replication ,Helicase ,DNA ,Molecular biology ,Branch migration ,Protein Structure, Tertiary ,enzymes and coenzymes (carbohydrates) ,biology.protein ,General Agricultural and Biological Sciences ,DNA Damage ,Protein Binding - Abstract
SummaryThree (BLM, WRN, and RECQ4) of the five human RecQ helicases are linked to genetic disorders characterized by genomic instability, cancer, and accelerated aging [1]. RECQ1, the first human RecQ helicase discovered [2–4] and the most abundant [5], was recently implicated in breast cancer [6, 7]. RECQ1 is an ATP-dependent DNA-unwinding enzyme (helicase) [8, 9] with roles in replication [10–12] and DNA repair [13–16]. RECQ1 is highly expressed in various tumors and cancer cell lines (for review, see [17]), and its suppression reduces cancer cell proliferation [14], suggesting a target for anti-cancer drugs. RECQ1’s assembly state plays a critical role in modulating its helicase, branch migration (BM), or strand annealing [18, 19]. The crystal structure of truncated RECQ1 [20, 21] resembles that of E. coli RecQ [22] with two RecA-like domains, a RecQ-specific zinc-binding domain and a winged-helix domain, the latter implicated in DNA strand separation and oligomer formation. In addition, a conserved aromatic loop (AL) is important for DNA unwinding by bacterial RecQ [23, 24] and truncated RECQ1 helicases [21]. To better understand the roles of RECQ1, two AL mutants (W227A and F231A) in full-length RECQ1 were characterized biochemically and genetically. The RECQ1 mutants were defective in helicase or BM but retained DNA binding, oligomerization, ATPase, and strand annealing. RECQ1-depleted HeLa cells expressing either AL mutant displayed reduced replication tract length, elevated dormant origin firing, and increased double-strand breaks that could be suppressed by exogenously expressed replication protein A (RPA). Thus, RECQ1 governs RPA’s availability in order to maintain normal replication dynamics, suppress DNA damage, and preserve genome homeostasis.
- Published
- 2015
7. Protein Degradation Pathways Regulate the Functions of Helicases in the DNA Damage Response and Maintenance of Genomic Stability
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Joshua A. Sommers, Robert M. Brosh, and Avvaru N. Suhasini
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DNA re-replication ,Proteasome Endopeptidase Complex ,congenital, hereditary, and neonatal diseases and abnormalities ,DNA Repair ,DNA repair ,lcsh:QR1-502 ,Eukaryotic DNA replication ,Review ,DNA damage response ,Biochemistry ,Genomic Instability ,lcsh:Microbiology ,Control of chromosome duplication ,ubiquitin ,Postreplication repair ,Animals ,Humans ,FANCM ,Cockayne syndrome ,Molecular Biology ,Replication protein A ,acetylation ,Genetics ,Werner syndrome ,biology ,Bloom’s syndrome ,phosphorylation ,DNA Helicases ,Genetic Diseases, Inborn ,Helicase ,nutritional and metabolic diseases ,helicase ,proteasome ,Fanconi Anemia ,post-translational modification ,Proteolysis ,biology.protein ,DNA Damage - Abstract
Degradation of helicases or helicase-like proteins, often mediated by ubiquitin-proteasomal pathways, plays important regulatory roles in cellular mechanisms that respond to DNA damage or replication stress. The Bloom's syndrome helicase (BLM) provides an example of how helicase degradation pathways, regulated by post-translational modifications and protein interactions with components of the Fanconi Anemia (FA) interstrand cross-link (ICL) repair pathway, influence cell cycle checkpoints, DNA repair, and replication restart. The FANCM DNA translocase can be targeted by checkpoint kinases that exert dramatic effects on FANCM stability and chromosomal integrity. Other work provides evidence that degradation of the F-box DNA helicase (FBH1) helps to balance translesion synthesis (TLS) and homologous recombination (HR) repair at blocked replication forks. Degradation of the helicase-like transcription factor (HLTF), a DNA translocase and ubiquitylating enzyme, influences the choice of post replication repair (PRR) pathway. Stability of the Werner syndrome helicase-nuclease (WRN) involved in the replication stress response is regulated by its acetylation. Turning to transcription, stability of the Cockayne Syndrome Group B DNA translocase (CSB) implicated in transcription-coupled repair (TCR) is regulated by a CSA ubiquitin ligase complex enabling recovery of RNA synthesis. Collectively, these studies demonstrate that helicases can be targeted for degradation to maintain genome homeostasis.
- Published
- 2015
8. Molecular functions and cellular roles of the ChlR1 (DDX11) helicase defective in the rare cohesinopathy Warsaw breakage syndrome
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Robert M. Brosh, Sanjay Kumar Bharti, Taraswi Banerjee, Yuliang Wu, Joshua A. Sommers, and Irfan Khan
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Genome instability ,DNA repair ,Mitomycin ,medicine.disease_cause ,Genomic Instability ,Article ,Substrate Specificity ,CHL1 ,DEAD-box RNA Helicases ,Cellular and Molecular Neuroscience ,DDX11 ,Neoplasms ,medicine ,Homeostasis ,Humans ,Abnormalities, Multiple ,Papillomaviridae ,Molecular Biology ,Gene ,Pharmacology ,Genetics ,Mutation ,biology ,DNA Breaks ,DNA Helicases ,Helicase ,Syndrome ,Cell Biology ,G-Quadruplexes ,Establishment of sister chromatid cohesion ,Phenotype ,biology.protein ,Molecular Medicine - Abstract
In 2010, a new recessive cohesinopathy disorder, designated Warsaw breakage syndrome (WABS), was described. The individual with WABS displayed microcephaly, pre- and postnatal growth retardation, and abnormal skin pigmentation. Cytogenetic analysis revealed mitomycin C (MMC)-induced chromosomal breakage; however, an additional sister chromatid cohesion defect was also observed. WABS is genetically linked to bi-allelic mutations in the ChlR1/DDX11 gene which encodes a protein of the conserved family of Iron–Sulfur (Fe–S) cluster DNA helicases. Mutations in the budding yeast ortholog of ChlR1, known as Chl1, were known to cause sister chromatid cohesion defects, indicating a conserved function of the gene. In 2012, three affected siblings were identified with similar symptoms to the original WABS case, and found to have a homozygous mutation in the conserved Fe–S domain of ChlR1, confirming the genetic linkage. Significantly, the clinically relevant mutations perturbed ChlR1 DNA unwinding activity. In addition to its genetic importance in human disease, ChlR1 is implicated in papillomavirus genome maintenance and cancer. Although its precise functions in genome homeostasis are still not well understood, ongoing molecular studies of ChlR1 suggest the helicase plays a critically important role in cellular replication and/or DNA repair.
- Published
- 2014
9. DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase
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Jun Zhou, Daniel L. Kaplan, Joshua A. Sommers, Johannes N. Spelbrink, Robert M. Brosh, Sanjay Kumar Bharti, and Jean-Louis Mergny
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DNA Replication ,Aging ,Mitochondrial DNA ,Ultraviolet Rays ,Base pair ,Molecular Sequence Data ,DNA and Chromosomes ,Biology ,Nucleic Acid Denaturation ,MT-RNR1 ,DNA, Mitochondrial ,Biochemistry ,Human mitochondrial genetics ,Substrate Specificity ,Evolution, Molecular ,Mitochondrial Proteins ,Neoplasms ,Animals ,Humans ,Disease ,Nucleotide Motifs ,Molecular Biology ,Conserved Sequence ,Sequence Deletion ,Genetics ,Homoplasmy ,Base Sequence ,Circular Dichroism ,DNA Helicases ,DNA replication ,Computational Biology ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,Cell Biology ,Telomere ,Recombinant Proteins ,Mitochondria ,G-Quadruplexes ,Genome, Mitochondrial ,DNAJA3 ,DNA Damage ,Mitochondrial DNA replication - Abstract
Contains fulltext : 138677.pdf (Publisher’s version ) (Open Access) Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the "Pattern Finder" G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase.
- Published
- 2014
10. Werner Syndrome Helicase Has a Critical Role in DNA Damage Responses in the Absence of a Functional Fanconi Anemia Pathway
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Monika Aggarwal, Chiara Iannascoli, Taraswi Banerjee, Pietro Pichierri, Joshua A. Sommers, Robert M. Brosh, and Robert H. Shoemaker
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DNA Replication ,Alkylating Agents ,congenital, hereditary, and neonatal diseases and abnormalities ,Cancer Research ,Werner Syndrome Helicase ,Fanconi anemia, complementation group C ,DNA Repair ,DNA damage ,Mitomycin ,Blotting, Western ,RAD51 ,Apoptosis ,Ataxia Telangiectasia Mutated Proteins ,DNA-Activated Protein Kinase ,Article ,Maleimides ,Fanconi anemia ,Chromosomal Instability ,medicine ,Humans ,DNA Breaks, Double-Stranded ,Enzyme Inhibitors ,RNA, Small Interfering ,education ,Cell Proliferation ,Werner syndrome ,education.field_of_study ,RecQ Helicases ,biology ,Nuclear Proteins ,nutritional and metabolic diseases ,Helicase ,Drug Synergism ,HCT116 Cells ,medicine.disease ,Molecular biology ,Chromatin ,enzymes and coenzymes (carbohydrates) ,Exodeoxyribonucleases ,Fanconi Anemia ,Oncology ,biology.protein ,Cancer research ,Drug Therapy, Combination ,Rad51 Recombinase ,Homologous recombination ,HeLa Cells - Abstract
Werner syndrome is genetically linked to mutations in WRN that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small-molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi anemia, a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2−/− cells, NSC 617145 acted synergistically with very low concentrations of mitomycin C to inhibit proliferation in a WRN-dependent manner and induce double-strand breaks (DSB) and chromosomal abnormalities. Under these conditions, ataxia–telangiectasia mutated activation and accumulation of DNA-dependent protein kinase, catalytic subunit pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2−/− cells exposed to NSC 617145 and mitomycin C, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination at ICL-induced DSBs. Thus, when the Fanconi anemia pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of Fanconi anemia–deficient tumors by their sensitization to DNA cross-linking agents is discussed. Cancer Res; 73(17); 5497–507. ©2013 AACR.
- Published
- 2013
11. Fanconi Anemia Group J Helicase and MRE11 Nuclease Interact To Facilitate the DNA Damage Response
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Jean-Yves Masson, Michael M. Seidman, Parameswary A. Muniandy, Robert M. Brosh, Joshua A. Sommers, Yan Coulombe, Avvaru N. Suhasini, and Sharon B. Cantor
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Exonuclease ,DNA Repair ,DNA repair ,DNA damage ,RAD51 ,Biology ,Fanconi anemia ,Chromosomal Instability ,Radiation, Ionizing ,medicine ,Humans ,DNA Breaks, Double-Stranded ,Molecular Biology ,MRE11 Homologue Protein ,Nuclease ,Endodeoxyribonucleases ,Ficusin ,Nuclear Proteins ,Recombinational DNA Repair ,Helicase ,Articles ,Cell Biology ,medicine.disease ,Molecular biology ,Fanconi Anemia Complementation Group Proteins ,Acid Anhydride Hydrolases ,Cell biology ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,Basic-Leucine Zipper Transcription Factors ,DNA Repair Enzymes ,biology.protein ,Carrier Proteins ,Homologous recombination ,DNA Damage ,HeLa Cells - Abstract
FANCJ mutations are linked to Fanconi anemia (FA) and increase breast cancer risk. FANCJ encodes a DNA helicase implicated in homologous recombination (HR) repair of double-strand breaks (DSBs) and interstrand cross-links (ICLs), but its mechanism of action is not well understood. Here we show with live-cell imaging that FANCJ recruitment to laser-induced DSBs but not psoralen-induced ICLs is dependent on nuclease-active MRE11. FANCJ interacts directly with MRE11 and inhibits its exonuclease activity in a specific manner, suggesting that FANCJ regulates the MRE11 nuclease to facilitate DSB processing and appropriate end resection. Cells deficient in FANCJ and MRE11 show increased ionizing radiation (IR) resistance, reduced numbers of γH2AX and RAD51 foci, and elevated numbers of DNA-dependent protein kinase catalytic subunit foci, suggesting that HR is compromised and the nonhomologous end-joining (NHEJ) pathway is elicited to help cells cope with IR-induced strand breaks. Interplay between FANCJ and MRE11 ensures a normal response to IR-induced DSBs, whereas FANCJ involvement in ICL repair is regulated by MLH1 and the FA pathway. Our findings are discussed in light of the current model for HR repair.
- Published
- 2013
12. Biochemical and Cell Biological Assays to Identify and Characterize DNA Helicase Inhibitors
- Author
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Taraswi Banerjee, Monika Aggarwal, Joshua A. Sommers, and Robert M. Brosh
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0301 basic medicine ,Premature aging ,DNA Replication ,DNA Repair ,DNA repair ,Cellular homeostasis ,Computational biology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Transcription (biology) ,medicine ,Humans ,Enzyme Inhibitors ,Molecular Biology ,Werner syndrome ,Genetics ,DNA replication ,DNA Helicases ,Helicase ,medicine.disease ,030104 developmental biology ,chemistry ,biology.protein ,Biological Assay ,DNA - Abstract
The growing number of DNA helicases implicated in hereditary disorders and cancer indicates that this particular class of enzymes plays key roles in genomic stability and cellular homeostasis. Indeed, a large body of work has provided molecular and cellular evidence that helicases act upon a variety of nucleic acid substrates and interact with numerous proteins to enact their functions in replication, DNA repair, recombination, and transcription. Understanding how helicases operate in unique and overlapping pathways is a great challenge to researchers. In this review, we describe a series of experimental approaches and methodologies to identify and characterize DNA helicase inhibitors which collectively provide an alternative and useful strategy to explore their biological significance in cell-based systems. These procedures were used in the discovery of biologically active compounds that inhibited the DNA unwinding function catalyzed by the human WRN helicase-nuclease defective in the premature aging disorder Werner syndrome. We describe in vitro and in vivo experimental approaches to characterize helicase inhibitors with WRN as the model, anticipating that these approaches may be extrapolated to other DNA helicases, particularly those implicated in DNA repair and/or the replication stress response.
- Published
- 2016
13. DNA Repair and Replication Fork Helicases Are Differentially Affected by Alkyl Phosphotriester Lesion
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Daniel L. Kaplan, Zvi Kelman, Yuliang Wu, Stephen Yu, Ting Xu, Avvaru N. Suhasini, Robert M. Brosh, and Joshua A. Sommers
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DNA Replication ,DNA, Bacterial ,DNA Repair ,DNA repair ,DNA polymerase II ,Eukaryotic DNA replication ,DNA and Chromosomes ,Biochemistry ,Catalysis ,Bacterial Proteins ,Escherichia coli ,Humans ,Molecular Biology ,Replication protein A ,chemistry.chemical_classification ,DNA ligase ,DNA clamp ,biology ,Methanobacterium ,DNA Helicases ,Helicase ,Cell Biology ,Organophosphates ,Models, Chemical ,chemistry ,biology.protein ,DNA supercoil ,lipids (amino acids, peptides, and proteins) - Abstract
DNA helicases are directly responsible for catalytically unwinding duplex DNA in an ATP-dependent and directionally specific manner and play essential roles in cellular nucleic acid metabolism. It has been conventionally thought that DNA helicases are inhibited by bulky covalent DNA adducts in a strand-specific manner. However, the effects of highly stable alkyl phosphotriester (PTE) lesions that are induced by chemical mutagens and refractory to DNA repair have not been previously studied for their effects on helicases. In this study, DNA repair and replication helicases were examined for unwinding a forked duplex DNA substrate harboring a single isopropyl PTE specifically positioned in the helicase-translocating or -nontranslocating strand within the double-stranded region. A comparison of SF2 helicases (RecQ, RECQ1, WRN, BLM, FANCJ, and ChlR1) with a SF1 DNA repair helicase (UvrD) and two replicative helicases (MCM and DnaB) demonstrates unique differences in the effect of the PTE on the DNA unwinding reactions catalyzed by these enzymes. All of the SF2 helicases tested were inhibited by the PTE lesion, whereas UvrD and the replication fork helicases were fully tolerant of the isopropyl backbone modification, irrespective of strand. Sequestration studies demonstrated that RECQ1 helicase was trapped by the PTE lesion only when it resided in the helicase-translocating strand. Our results are discussed in light of the current models for DNA unwinding by helicases that are likely to encounter sugar phosphate backbone damage during biological DNA transactions.
- Published
- 2012
14. Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom's syndrome
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Robert M. Brosh, Avvaru N. Suhasini, Sudha Sharma, Ian D. Hickson, Phillip S North, Sharon B. Cantor, Nina A Rawtani, Yuliang Wu, Joshua A. Sommers, and Georgina Mosedale
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Genome instability ,congenital, hereditary, and neonatal diseases and abnormalities ,General Immunology and Microbiology ,biology ,urogenital system ,General Neuroscience ,DNA replication ,nutritional and metabolic diseases ,Helicase ,medicine.disease ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,chemistry ,Fanconi anemia ,Chromosome instability ,medicine ,biology.protein ,Bloom syndrome ,Molecular Biology ,Gene ,DNA - Abstract
Bloom’s syndrome (BS) and Fanconi anemia (FA) are autosomal recessive disorders characterized by cancer and chromosomal instability. BS and FA group J arise from mutations in the BLM and FANCJ genes, respectively, which encode DNA helicases. In this work, FANCJ and BLM were found to interact physically and functionally in human cells and co-localize to nuclear foci in response to replication stress. The cellular level of BLM is strongly dependent upon FANCJ, and BLM is degraded by a proteasome-mediated pathway when FANCJ is depleted. FANCJdeficient cells display increased sister chromatid exchange and sensitivity to replication stress. Expression of a FANCJ C-terminal fragment that interacts with BLM exerted a dominant negative effect on hydroxyurea resistance by interfering with the FANCJ–BLM interaction. FANCJ and BLM synergistically unwound a DNA duplex substrate with sugar phosphate backbone discontinuity, but not an ‘undamaged’ duplex. Collectively, the results suggest that FANCJ catalytic activity and its effect on BLM protein stability contribute to preservation of genomic stability and a normal response to replication stress.
- Published
- 2011
15. Inhibition of helicase activity by a small molecule impairs Werner syndrome helicase (WRN) function in the cellular response to DNA damage or replication stress
- Author
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Robert M. Brosh, Monika Aggarwal, Robert H. Shoemaker, and Joshua A. Sommers
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DNA Replication ,congenital, hereditary, and neonatal diseases and abnormalities ,Time Factors ,Werner Syndrome Helicase ,Cell Survival ,DNA damage ,DNA repair ,Immunoblotting ,Apoptosis ,Topoisomerase-I Inhibitor ,Cell Line ,S Phase ,Histones ,Maleimides ,Stress, Physiological ,Cell Line, Tumor ,Proliferating Cell Nuclear Antigen ,medicine ,Humans ,Bloom syndrome ,Enzyme Inhibitors ,education ,Oxazoles ,dnaB helicase ,Cell Proliferation ,Adenosine Triphosphatases ,education.field_of_study ,Multidisciplinary ,Dose-Response Relationship, Drug ,Molecular Structure ,RecQ Helicases ,biology ,nutritional and metabolic diseases ,Helicase ,Drug Synergism ,Biological Sciences ,medicine.disease ,Molecular biology ,Cell biology ,Proliferating cell nuclear antigen ,Exodeoxyribonucleases ,biology.protein ,Topoisomerase I Inhibitors ,Topotecan ,DNA Damage ,HeLa Cells - Abstract
Modulation of DNA repair proteins by small molecules has attracted great interest. An in vitro helicase activity screen was used to identify molecules that modulate DNA unwinding by Werner syndrome helicase (WRN), mutated in the premature aging disorder Werner syndrome. A small molecule from the National Cancer Institute Diversity Set designated NSC 19630 [1-(propoxymethyl)-maleimide] was identified that inhibited WRN helicase activity but did not affect other DNA helicases [Bloom syndrome (BLM), Fanconi anemia group J (FANCJ), RECQ1, RecQ, UvrD, or DnaB). Exposure of human cells to NSC 19630 dramatically impaired growth and proliferation, induced apoptosis in a WRN-dependent manner, and resulted in elevated γ-H2AX and proliferating cell nuclear antigen (PCNA) foci. NSC 19630 exposure led to delayed S-phase progression, consistent with the accumulation of stalled replication forks, and to DNA damage in a WRN-dependent manner. Exposure to NSC 19630 sensitized cancer cells to the G-quadruplex–binding compound telomestatin or a poly(ADP ribose) polymerase (PARP) inhibitor. Sublethal dosage of NSC 19630 and the chemotherapy drug topotecan acted synergistically to inhibit cell proliferation and induce DNA damage. The use of this WRN helicase inhibitor molecule may provide insight into the importance of WRN-mediated pathway(s) important for DNA repair and the replicational stress response.
- Published
- 2011
16. Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes
- Author
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Joshua A. Sommers, Alexander V. Mazin, Yuliang Wu, Avvaru N. Suhasini, Thomas A. Leonard, Kazuo Shin-ya, Robert M. Brosh, Hiroyuki Kitao, and Julianna S. Deakyne
- Subjects
DNA Repair ,HMG-box ,Hematopoiesis and Stem Cells ,DNA repair ,Iron ,Mitomycin ,Molecular Sequence Data ,Immunology ,Mutation, Missense ,In Vitro Techniques ,Biochemistry ,law.invention ,chemistry.chemical_compound ,Adenosine Triphosphate ,Mutant protein ,Fanconi anemia ,law ,medicine ,Humans ,Amino Acid Sequence ,Oxazoles ,Adenosine Triphosphatases ,biology ,Protein Stability ,DNA Helicases ,Helicase ,DNA ,Cell Biology ,Hematology ,medicine.disease ,Molecular biology ,Fanconi Anemia Complementation Group Proteins ,Recombinant Proteins ,Basic-Leucine Zipper Transcription Factors ,Amino Acid Substitution ,chemistry ,biology.protein ,Recombinant DNA ,Mutant Proteins ,Homologous recombination - Abstract
Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and susceptibility to leukemia and other cancers. FANCJ, one of 13 genes linked to FA, encodes a DNA helicase proposed to operate in homologous recombination repair and replicational stress response. The pathogenic FANCJ-A349P amino acid substitution resides immediately adjacent to a highly conserved cysteine of the iron-sulfur domain. Given the genetic linkage of the FANCJ-A349P allele to FA, we investigated the effect of this particular mutation on the biochemical and cellular functions of the FANCJ protein. Purified recombinant FANCJ-A349P protein had reduced iron and was defective in coupling adenosine triphosphate (ATP) hydrolysis and translocase activity to unwinding forked duplex or G-quadruplex DNA substrates or disrupting protein-DNA complexes. The FANCJ-A349P allele failed to rescue cisplatin or telomestatin sensitivity of a FA-J null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, expression of FANCJ-A349P in a wild-type background exerted a dominant-negative effect, indicating that the mutant protein interferes with normal DNA metabolism. The ability of FANCJ to use the energy from ATP hydrolysis to produce the force required to unwind DNA or destabilize protein bound to DNA is required for its role in DNA repair.
- Published
- 2010
17. Delineation of WRN helicase function with EXO1 in the replicational stress response
- Author
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Robert M. Brosh, Joshua A. Sommers, Monika Aggarwal, and Christa Morris
- Subjects
DNA Replication ,Premature aging ,Genome instability ,congenital, hereditary, and neonatal diseases and abnormalities ,Saccharomyces cerevisiae Proteins ,Werner Syndrome Helicase ,DNA Repair ,DNA repair ,Saccharomyces cerevisiae ,Biology ,Biochemistry ,Article ,Drug Resistance, Fungal ,Stress, Physiological ,medicine ,Humans ,education ,Molecular Biology ,Werner syndrome ,education.field_of_study ,RecQ Helicases ,Genetic Complementation Test ,Fungal genetics ,nutritional and metabolic diseases ,Helicase ,Cell Biology ,Methyl Methanesulfonate ,medicine.disease ,Molecular biology ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,DNA Repair Enzymes ,Exodeoxyribonucleases ,biology.protein ,Homologous recombination - Abstract
The WRN gene defective in the premature aging disorder Werner syndrome encodes a helicase/exonuclease. We examined the ability of WRN to rescue DNA damage sensitivity of a yeast mutant defective in the Rad50 subunit of Mre11-Rad50- Xrs2 nuclease complex implicated in homologous recombination repair. Genetic studies revealed WRN operates in a yEXO1-dependent pathway to rescue rad50 sensitivity to methylmethane sulfonate (MMS) and prevent mitotic catastrophe. WRN helicase, but not exonuclease, is required for MMS resistance. WRN missense mutations in helicase or RecQ C-terminal domains interfered with the ability of WRN to rescue rad50 MMS sensitivity. WRN does not rescue rad50 ionizing radiation (IR) sensitivity, suggesting that WRN, in collaboration with yEXO1, is tailored to relieve replicational stress imposed by alkylated base damage. WRN and yEXO1 are associated with each other in vivo. Purified WRN stimulates hEXO1 nuclease activity on DNA substrates associated with a stalled or regressed replication fork. We propose WRN helicase operates in an EXO1-dependent pathway to help cells survive replicational stress. In contrast to WRN, BLM helicase defective in Bloom’s syndrome failed to rescue rad50 MMS sensitivity, but partially restored IR resistance, suggesting a delineation of function by the human RecQ helicases.
- Published
- 2010
18. Close encounters for the first time: Helicase interactions with DNA damage
- Author
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Joshua A. Sommers, Robert M. Brosh, and Irfan Khan
- Subjects
biology ,DNA repair ,DNA damage ,Circular bacterial chromosome ,DNA Helicases ,Helicase ,Eukaryotic DNA replication ,Cell Biology ,Biochemistry ,Article ,Substrate Specificity ,Control of chromosome duplication ,biology.protein ,Biophysics ,DNA supercoil ,Molecular Biology ,Replication protein A ,DNA Damage - Abstract
DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism.
- Published
- 2015
19. p53 Modulates RPA-Dependent and RPA-Independent WRN Helicase Activity
- Author
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Kevin M. Doherty, Robert M. Brosh, Parimal Karmakar, Joshua A. Sommers, Mark K. Kenny, Qin Yang, Curtis C. Harris, and Sudha Sharma
- Subjects
DNA Replication ,Genome instability ,congenital, hereditary, and neonatal diseases and abnormalities ,Cancer Research ,Werner Syndrome Helicase ,DNA Repair ,Immunoprecipitation ,chemistry.chemical_compound ,Replication Protein A ,medicine ,Humans ,education ,Replication protein A ,Werner syndrome ,education.field_of_study ,RecQ Helicases ,biology ,DNA Helicases ,nutritional and metabolic diseases ,Helicase ,DNA ,Fibroblasts ,Telomere ,medicine.disease ,Molecular biology ,RNA Helicase A ,Protein Structure, Tertiary ,DNA-Binding Proteins ,Exodeoxyribonucleases ,Oncology ,chemistry ,biology.protein ,Werner Syndrome ,Tumor Suppressor Protein p53 - Abstract
Werner syndrome is a hereditary disorder characterized by the early onset of age-related symptoms, including cancer. The absence of a p53-WRN helicase interaction may disrupt the signal to direct S-phase cells into apoptosis for programmed cell death and contribute to the pronounced genomic instability and cancer predisposition in Werner syndrome cells. Results from coimmunoprecipitation studies indicate that WRN is associated with replication protein A (RPA) and p53 in vivo before and after treatment with the replication inhibitor hydroxyurea or γ-irradiation that introduces DNA strand breaks. Analysis of the protein interactions among purified recombinant WRN, RPA, and p53 proteins indicate that all three protein pairs bind with similar affinity in the low nanomolar range. In vitro studies show that p53 inhibits RPA-stimulated WRN helicase activity on an 849-bp M13 partial duplex substrate. p53 also inhibited WRN unwinding of a short (19-bp) forked duplex substrate in the absence of RPA. WRN unwinding of the forked duplex substrate was specific, because helicase inhibition mediated by p53 was retained in the presence of excess competitor DNA and was significantly reduced or absent in helicase reactions catalyzed by a WRN helicase domain fragment lacking the p53 binding site or the human RECQ1 DNA helicase, respectively. p53 effectively inhibited WRN helicase activity on model DNA substrate intermediates of replication/repair, a 5′ ssDNA flap structure and a synthetic replication fork. Regulation of WRN helicase activity by p53 is likely to play an important role in genomic integrity surveillance, a vital function in the prevention of tumor progression.
- Published
- 2005
20. Biochemical and Kinetic Characterization of the DNA Helicase and Exonuclease Activities of Werner Syndrome Protein
- Author
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Joshua A. Sommers, Robert M. Brosh, and Saba Choudhary
- Subjects
Exonucleases ,Genome instability ,Exonuclease ,Premature aging ,congenital, hereditary, and neonatal diseases and abnormalities ,Insecta ,Time Factors ,Werner Syndrome Helicase ,Iron ,DNA, Single-Stranded ,Biochemistry ,Catalysis ,Cofactor ,Cell Line ,chemistry.chemical_compound ,Adenosine Triphosphate ,Nickel ,Fluorescence Resonance Energy Transfer ,Animals ,Humans ,Magnesium ,Binding site ,education ,Molecular Biology ,Adenosine Triphosphatases ,Ions ,Manganese ,education.field_of_study ,Dose-Response Relationship, Drug ,RecQ Helicases ,biology ,DNA Helicases ,nutritional and metabolic diseases ,Helicase ,DNA ,Cell Biology ,Molecular biology ,Kinetics ,Exodeoxyribonucleases ,chemistry ,Metals ,biology.protein ,Copper - Abstract
The WRN gene, defective in the premature aging and genome instability disorder Werner syndrome, encodes a protein with DNA helicase and exonuclease activities. In this report, cofactor requirements for WRN catalytic activities were examined. WRN helicase performed optimally at an equimolar concentration (1 mm) of Mg(2+) and ATP with a K(m) of 140 microm for the ATP-Mg(2+) complex. The initial rate of WRN helicase activity displayed a hyperbolic dependence on ATP-Mg(2+) concentration. Mn(2+) and Ni(2+) substituted for Mg(2+) as a cofactor for WRN helicase, whereas Fe(2+) or Cu(2+) (10 microm) profoundly inhibited WRN unwinding in the presence of Mg(2+).Zn(2+) (100 microm) was preferred over Mg(2+) as a metal cofactor for WRN exonuclease activity and acts as a molecular switch, converting WRN from a helicase to an exonuclease. Zn(2+) strongly stimulated the exonuclease activity of a WRN exonuclease domain fragment, suggesting a Zn(2+) binding site in the WRN exonuclease domain. A fluorometric assay was used to study WRN helicase kinetics. The initial rate of unwinding increased with WRN concentration, indicating that excess enzyme over DNA substrate improved the ability of WRN to unwind the DNA substrate. Under presteady state conditions, the burst amplitude revealed a 1:1 ratio between WRN and DNA substrate, suggesting an active monomeric form of the helicase. These are the first reported kinetic parameters of a human RecQ unwinding reaction based on real time measurements, and they provide mechanistic insights into WRN-catalyzed DNA unwinding.
- Published
- 2004
21. Stimulation of Flap Endonuclease-1 by the Bloom's Syndrome Protein
- Author
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Joshua A. Sommers, Robert M. Brosh, Vilhelm A. Bohr, Ian D. Hickson, Leonard Wu, and Sudha Sharma
- Subjects
DNA Replication ,Genome instability ,congenital, hereditary, and neonatal diseases and abnormalities ,Saccharomyces cerevisiae Proteins ,Time Factors ,Werner Syndrome Helicase ,DNA Repair ,Flap Endonucleases ,DNA repair ,RecQ helicase ,Oligonucleotides ,Flap structure-specific endonuclease 1 ,Enzyme-Linked Immunosorbent Assay ,Saccharomyces cerevisiae ,Biology ,Biochemistry ,Catalysis ,Humans ,Flap endonuclease ,education ,Molecular Biology ,Adenosine Triphosphatases ,Cell Nucleus ,Recombination, Genetic ,education.field_of_study ,Endodeoxyribonucleases ,Dose-Response Relationship, Drug ,Models, Genetic ,RecQ Helicases ,Okazaki fragments ,urogenital system ,DNA Helicases ,DNA replication ,nutritional and metabolic diseases ,DNA ,Cell Biology ,Precipitin Tests ,Molecular biology ,Recombinant Proteins ,DNA-Binding Proteins ,Kinetics ,Exodeoxyribonucleases ,Amylose ,HeLa Cells ,Protein Binding - Abstract
Bloom's syndrome (BS) is a rare autosomal recessive genetic disorder associated with genomic instability and an elevated risk of cancer. Cellular features of BS include an accumulation of abnormal replication intermediates and increased sister chromatid exchange. Although it has been suggested that the underlying defect responsible for hyper-recombination in BS cells is a temporal delay in the maturation of DNA replication intermediates, the precise role of the BS gene product, BLM, in DNA metabolism remains elusive. We report here a novel interaction of the BLM protein with the human 5'-flap endonuclease/5'-3' exonuclease (FEN-1), a genome stability factor involved in Okazaki fragment processing and DNA repair. BLM protein stimulates both the endonucleolytic and exonucleolytic cleavage activity of FEN-1 and this functional interaction is independent of BLM catalytic activity. BLM and FEN-1 are associated with each other in human nuclei as shown by their reciprocal co-immunoprecipitation from HeLa nuclear extracts. The BLM-FEN-1 physical interaction is mediated through a region of the BLM C-terminal domain that shares homology with the FEN-1 interaction domain of the Werner syndrome protein, a RecQ helicase family member homologous to BLM. This study provides the first evidence for a direct interaction of BLM with a human nucleolytic enzyme. We suggest that functional interactions between RecQ helicases and Rad2 family nucleases serve to process DNA substrates that are intermediates in DNA replication and repair.
- Published
- 2004
22. The Exonucleolytic and Endonucleolytic Cleavage Activities of Human Exonuclease 1 Are Stimulated by an Interaction with the Carboxyl-terminal Region of the Werner Syndrome Protein
- Author
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Teresa M. Wilson, Sudha Sharma, Robert M. Brosh, Henry C. Driscoll, Joshua A. Sommers, and Laura A. Uzdilla
- Subjects
Genome instability ,congenital, hereditary, and neonatal diseases and abnormalities ,Werner Syndrome Helicase ,Flap Endonucleases ,Flap structure-specific endonuclease 1 ,DNA, Single-Stranded ,Biology ,Cleavage (embryo) ,Biochemistry ,law.invention ,Exonuclease 1 ,Werner Syndrome Protein ,law ,Cleave ,Humans ,Flap endonuclease ,Molecular Biology ,Binding Sites ,Endodeoxyribonucleases ,Dose-Response Relationship, Drug ,RecQ Helicases ,fungi ,DNA Helicases ,nutritional and metabolic diseases ,Cell Biology ,Precipitin Tests ,Protein Structure, Tertiary ,carbohydrates (lipids) ,Kinetics ,DNA Repair Enzymes ,Exodeoxyribonucleases ,Recombinant DNA ,Protein Binding - Abstract
Exonuclease 1 (EXO-1), a member of the RAD2 family of nucleases, has recently been proposed to function in the genetic pathways of DNA recombination, repair, and replication which are important for genome integrity. Although the role of EXO-1 is not well understood, its 5' to 3'-exonuclease and flap endonuclease activities may cleave intermediates that arise during DNA metabolism. In this study, we provide evidence that the Werner syndrome protein (WRN) physically interacts with human EXO-1 and dramatically stimulates both the exonucleolytic and endonucleolytic incision functions of EXO-1. The functional interaction between WRN and EXO-1 is mediated by a protein domain of WRN which interacts with flap endonuclease 1 (FEN-1). Thus, the genomic instability observed in WRN-/- cells may be at least partially attributed to the lack of interactions between the WRN protein and human nucleases including EXO-1.
- Published
- 2003
23. Biochemical Characterization of the DNA Substrate Specificity of Werner Syndrome Helicase
- Author
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Joshua A. Sommers, Robert M. Brosh, and Juwaria F. Waheed
- Subjects
DNA Replication ,Premature aging ,Exonuclease ,Genome instability ,congenital, hereditary, and neonatal diseases and abnormalities ,Werner Syndrome Helicase ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Humans ,education ,Molecular Biology ,education.field_of_study ,RecQ Helicases ,biology ,Okazaki fragments ,DNA Helicases ,DNA replication ,nutritional and metabolic diseases ,Helicase ,DNA ,Cell Biology ,Molecular biology ,Cell biology ,Exodeoxyribonucleases ,chemistry ,biology.protein ,Werner Syndrome - Abstract
Werner syndrome is a hereditary premature aging disorder characterized by genome instability. The product of the gene defective in WS, WRN, is a helicase/exonuclease that presumably functions in DNA metabolism. To understand the DNA structures WRN acts upon in vivo, we examined its substrate preferences for unwinding. WRN unwound a 3'-single-stranded (ss)DNA-tailed duplex substrate with streptavidin bound to the end of the 3'-ssDNA tail, suggesting that WRN does not require a free DNA end to unwind the duplex; however, WRN was completely blocked by streptavidin bound to the 3'-ssDNA tail 6 nucleotides upstream of the single-stranded/double-stranded DNA junction. WRN efficiently unwound the forked duplex with streptavidin bound just upstream of the junction, suggesting that WRN recognizes elements of the fork structure to initiate unwinding. WRN unwound two important intermediates of replication/repair, a 5'-ssDNA flap substrate and a synthetic replication fork. WRN was able to translocate on the lagging strand of the synthetic replication fork to unwind duplex ahead of the fork. For the 5'-flap structure, WRN specifically displaced the 5'-flap oligonucleotide, suggesting a role of WRN in Okazaki fragment processing. The ability of WRN to target DNA replication/repair intermediates may be relevant to its role in genome stability maintenance.
- Published
- 2002
24. Werner Protein Is a Target of DNA-dependent Protein Kinase in Vivo and in Vitro, and Its Catalytic Activities Are Regulated by Phosphorylation
- Author
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Susan P. Lees Miller, Parimal Karmakar, Robert M. Brosh, Wen-Hsing Cheng, Dale A. Ramsden, Jason Piotrowski, Vilhelm A. Bohr, Joshua A. Sommers, and Carey M. Snowden
- Subjects
Exonuclease ,congenital, hereditary, and neonatal diseases and abnormalities ,Insecta ,Werner Syndrome Helicase ,Protein subunit ,Phosphatase ,DNA-Activated Protein Kinase ,Protein Serine-Threonine Kinases ,Biochemistry ,Catalysis ,Catalytic Domain ,medicine ,Animals ,Humans ,Phosphorylation ,Kinase activity ,Protein kinase A ,Molecular Biology ,DNA Primers ,Werner syndrome ,Base Sequence ,RecQ Helicases ,biology ,DNA Helicases ,Nuclear Proteins ,nutritional and metabolic diseases ,Helicase ,Cell Biology ,medicine.disease ,Molecular biology ,Recombinant Proteins ,Cell biology ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,Exodeoxyribonucleases ,biology.protein ,Werner Syndrome ,DNA Damage ,HeLa Cells - Abstract
Human Werner Syndrome is characterized by early onset of aging, elevated chromosomal instability, and a high incidence of cancer. Werner protein (WRN) is a member of the recQ gene family, but unlike other members of the recQ family, it contains a unique 3'-->5' exonuclease activity. We have reported previously that human Ku heterodimer interacts physically with WRN and functionally stimulates WRN exonuclease activity. Because Ku and DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), form a complex at DNA ends, we have now explored the possibility of functional modulation of WRN exonuclease activity by DNA-PK. We find that although DNA-PKcs alone does not affect the WRN exonuclease activity, the additional presence of Ku mediates a marked inhibition of it. The inhibition of WRN exonuclease by DNA-PKcs requires the kinase activity of DNA-PKcs. WRN is a target for DNA-PKcs phosphorylation, and this phosphorylation requires the presence of Ku. We also find that treatment of recombinant WRN with a Ser/Thr phosphatase enhances WRN exonuclease and helicase activities and that WRN catalytic activity can be inhibited by rephosphorylation of WRN with DNA-PK. Thus, the level of phosphorylation of WRN appears to regulate its catalytic activities. WRN forms a complex, both in vitro and in vivo, with DNA-PKC. WRN is phosphorylated in vivo after treatment of cells with DNA-damaging agents in a pathway that requires DNA-PKcs. Thus, WRN protein is a target for DNA-PK phosphorylation in vitro and in vivo, and this phosphorylation may be a way of regulating its different catalytic activities, possibly in the repair of DNA dsb.
- Published
- 2002
25. Impact of age-associated cyclopurine lesions on DNA repair helicases
- Author
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Joshua A. Sommers, Taraswi Banerjee, Jochen Kuper, Avvaru N. Suhasini, Daniel L. Kaplan, Irfan Khan, Robert M. Brosh, and Caroline Kisker
- Subjects
DNA Repair ,DNA repair ,DNA damage ,Archaeal Proteins ,lcsh:Medicine ,DNA replication ,Biochemistry ,chemistry.chemical_compound ,DDX11 ,ddc:570 ,DNA metabolism ,Humans ,lcsh:Science ,Molecular Biology ,dnaB helicase ,Multidisciplinary ,Deoxyadenosines ,Biology and life sciences ,biology ,lcsh:R ,DNA Helicases ,Deoxyguanosine ,Helicase ,DNA ,Archaea ,Biochemical Activity ,Molecular biology ,Recombinant Proteins ,chemistry ,Molecular Machines ,biology.protein ,lcsh:Q ,Research Article ,Nucleotide excision repair - Abstract
8,5′ cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases (RECQ1, BLM, WRN, RecQ) were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand. SF2 Fe-S helicases (FANCJ, DDX11 (ChlR1), DinG, XPD) displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal Thermoplasma acidophilum XPD (taXPD), homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in the cellular response to DNA damage and DNA repair.
- Published
- 2014
26. Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity
- Author
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Parimal Karmakar, Patricia L. Opresko, Robert M. Brosh, Irina I. Dianova, Grigory L. Dianov, Cayetano von Kobbe, Vilhelm A. Bohr, Jason Piotrowski, and Joshua A. Sommers
- Subjects
Exonucleases ,Premature aging ,Genome instability ,congenital, hereditary, and neonatal diseases and abnormalities ,Werner Syndrome Helicase ,Flap Endonucleases ,Macromolecular Substances ,Recombinant Fusion Proteins ,Flap structure-specific endonuclease 1 ,Catalysis ,Article ,General Biochemistry, Genetics and Molecular Biology ,Proliferating Cell Nuclear Antigen ,Replication Protein A ,medicine ,Humans ,Flap endonuclease ,education ,Molecular Biology ,Werner syndrome ,Adenosine Triphosphatases ,education.field_of_study ,Endodeoxyribonucleases ,RecQ Helicases ,General Immunology and Microbiology ,biology ,General Neuroscience ,DNA Helicases ,DNA replication ,nutritional and metabolic diseases ,Helicase ,DNA ,medicine.disease ,Molecular biology ,Peptide Fragments ,Protein Structure, Tertiary ,DNA-Binding Proteins ,Enzyme Activation ,Exodeoxyribonucleases ,biology.protein ,Werner Syndrome ,HeLa Cells - Abstract
Werner syndrome (WS) is a human premature aging disorder characterized by chromosomal instability. The cellular defects of WS presumably reflect compromised or aberrant function of a DNA metabolic pathway that under normal circumstances confers stability to the genome. We report a novel interaction of the WRN gene product with the human 5′ flap endonuclease/5′–3′ exonuclease (FEN‐1), a DNA structure‐specific nuclease implicated in DNA replication, recombination and repair. WS protein (WRN) dramatically stimulates the rate of FEN‐1 cleavage of a 5′ flap DNA substrate. The WRN–FEN‐1 functional interaction is independent of WRN catalytic function and mediated by a 144 amino acid domain of WRN that shares homology with RecQ DNA helicases. A physical interaction between WRN and FEN‐1 is demonstrated by their co‐immunoprecipitation from HeLa cell lysate and affinity pull‐down experiments using a recombinant C‐terminal fragment of WRN. The underlying defect of WS is discussed in light of the evidence for the interaction between WRN and FEN‐1.
- Published
- 2001
27. Targeting an Achilles’ heel of cancer with a WRN helicase inhibitor
- Author
-
Joshua A. Sommers, Taraswi Banerjee, Robert M. Brosh, and Monika Aggarwal
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,DNA End-Joining Repair ,DNA repair ,DNA damage ,Mitomycin ,Synthetic lethality ,Biology ,Models, Biological ,Werner Syndrome Helicase ,Cell Line ,Maleimides ,Stress, Physiological ,Report ,Neoplasms ,FANCD2 ,medicine ,Humans ,Enzyme Inhibitors ,education ,Molecular Biology ,Werner syndrome ,education.field_of_study ,RecQ Helicases ,Helicase ,nutritional and metabolic diseases ,Cell Biology ,medicine.disease ,Molecular biology ,Fanconi Anemia ,Cancer research ,biology.protein ,Developmental Biology ,Signal Transduction - Abstract
Our recently published work suggests that DNA helicases such as the Werner syndrome helicase (WRN) represent a novel class of proteins to target for anticancer therapy. Specifically, pharmacological inhibition of WRN helicase activity in human cells defective in the Fanconi anemia (FA) pathway of interstrand cross-link (ICL) repair are sensitized to the DNA cross-linking agent and chemotherapy drug mitomycin C (MMC) by the WRN helicase inhibitor NSC 617145. (1) The mechanistic basis for the synergistic interaction between NSC 617145 and MMC is discussed in this paper and extrapolated to potential implications for genetic or chemically induced synthetic lethality provoked by cellular exposure to the WRN helicase inhibitor under the context of relevant DNA repair deficiencies associated with cancers or induced by small-molecule inhibitors. Experimental data are presented showing that small-molecule inhibition of WRN helicase elevates sensitivity to MMC-induced stress in human cells that are deficient in both FANCD2 and DNA protein kinase catalytic subunit (DNA-PKcs). These findings suggest a model in which drug-mediated inhibition of WRN helicase activity exacerbates the deleterious effects of MMC-induced DNA damage when both the FA and NHEJ pathways are defective. We conclude with a perspective for the FA pathway and synthetic lethality and implications for DNA repair helicase inhibitors that can be developed for anticancer strategies.
- Published
- 2013
28. Specialization among Iron-Sulfur Cluster Helicases to Resolve G-quadruplex DNA Structures That Threaten Genomic Stability*
- Author
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Kazuo Shin-ya, Caroline Kisker, Fourbears George, Marie-Paule Teulade-Fichou, Robert M. Brosh, Sanjay Kumar Bharti, Joshua A. Sommers, Jochen Kuper, and Florian Hamon
- Subjects
Genome instability ,DNA Replication ,Iron-Sulfur Proteins ,congenital, hereditary, and neonatal diseases and abnormalities ,Guanine ,DNA Repair ,DNA repair ,DNA damage ,Thermoplasma ,DNA and Chromosomes ,G-quadruplex ,Ligands ,Biochemistry ,Genomic Instability ,chemistry.chemical_compound ,Inhibitory Concentration 50 ,hemic and lymphatic diseases ,Escherichia coli ,Humans ,Molecular Biology ,Genetics ,biology ,DNA replication ,DNA Helicases ,Helicase ,nutritional and metabolic diseases ,Cell Biology ,DNA ,Fanconi Anemia Complementation Group Proteins ,Recombinant Proteins ,G-Quadruplexes ,Basic-Leucine Zipper Transcription Factors ,chemistry ,Gene Expression Regulation ,biology.protein ,RNA Interference ,Nucleotide excision repair - Abstract
G-quadruplex (G4) DNA, an alternate structure formed by Hoogsteen hydrogen bonds between guanines in G-rich sequences, threatens genomic stability by perturbing normal DNA transactions including replication, repair, and transcription. A variety of G4 topologies (intra- and intermolecular) can form in vitro, but the molecular architecture and cellular factors influencing G4 landscape in vivo are not clear. Helicases that unwind structured DNA molecules are emerging as an important class of G4-resolving enzymes. The BRCA1-associated FANCJ helicase is among those helicases able to unwind G4 DNA in vitro, and FANCJ mutations are associated with breast cancer and linked to Fanconi anemia. FANCJ belongs to a conserved iron-sulfur (Fe S) cluster family of helicases important for genomic stability including XPD (nucleotide excision repair), DDX11 (sister chromatid cohesion), and RTEL (telomere metabolism), genetically linked to xeroderma pigmentosum/Cockayne syndrome, Warsaw breakage syndrome, and dyskeratosis congenita, respectively. To elucidate the role of FANCJ in genomic stability, its molecular functions in G4 metabolism were examined. FANCJ efficiently unwound in a kinetic and ATPase-dependent manner entropically favored unimolecular G4 DNA, whereas other Fe-S helicases tested did not. The G4-specific ligands Phen-DC3 or Phen-DC6 inhibited FANCJ helicase on unimolecular G4 ∼1000-fold better than bi- or tetramolecular G4 DNA. The G4 ligand telomestatin induced DNA damage in human cells deficient in FANCJ but not DDX11 or XPD. These findings suggest FANCJ is a specialized Fe-S cluster helicase that preserves chromosomal stability by unwinding unimolecular G4 DNA likely to form in transiently unwound single-stranded genomic regions. Background: The Fe-S helicase FANCJ implicated in Fanconi anemia plays important roles in DNA replication and repair. Results: FANCJ, but not the Fe-S XPD or DDX11 helicases, unwinds unimolecular G4 DNA. Conclusion: FANCJ is a specialized Fe-S helicase, preventing G4-induced DNA damage. Significance: FANCJ has a unique role in DNA metabolism to prevent G4 accumulation that causes genomic instability.
- Published
- 2013
29. Human RECQ1 interacts with Ku70/80 and modulates DNA end-joining of double-strand breaks
- Author
-
Sudha Sharma, Alexei Stortchevoi, Robert M. Brosh, Joshua A. Sommers, and Swetha Parvathaneni
- Subjects
Genome instability ,DNA End-Joining Repair ,RecQ helicase ,Oligonucleotides ,lcsh:Medicine ,Electrophoretic Mobility Shift Assay ,Toxicology ,Biochemistry ,Molecular cell biology ,0302 clinical medicine ,DNA Breaks, Double-Stranded ,Bloom syndrome ,Protein Interaction Maps ,lcsh:Science ,0303 health sciences ,Multidisciplinary ,RecQ Helicases ,Chromosome Biology ,Antigens, Nuclear ,Enzymes ,Nucleic acids ,030220 oncology & carcinogenesis ,Protein Binding ,Research Article ,Premature aging ,congenital, hereditary, and neonatal diseases and abnormalities ,Genetic Toxicology ,DNA repair ,DNA damage ,Biology ,03 medical and health sciences ,DNA-binding proteins ,medicine ,Humans ,Ku Autoantigen ,030304 developmental biology ,lcsh:R ,Proteins ,Helicase ,nutritional and metabolic diseases ,DNA ,medicine.disease ,Molecular biology ,biology.protein ,Nucleic Acid Conformation ,lcsh:Q ,HeLa Cells - Abstract
Genomic instability is a known precursor to cancer and aging. The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in maintaining genome stability in all living organisms. Human RecQ homologs include RECQ1, BLM, WRN, RECQ4, and RECQ5β, three of which have been linked to diseases with elevated risk of cancer and growth defects (Bloom Syndrome and Rothmund-Thomson Syndrome) or premature aging (Werner Syndrome). RECQ1, the first RecQ helicase discovered and the most abundant in human cells, is the least well understood of the five human RecQ homologs. We have previously described that knockout of RECQ1 in mice or knockdown of its expression in human cells results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased load of DNA damage and heightened sensitivity to ionizing radiation. We have now obtained evidence implicating RECQ1 in the nonhomologous end-joining pathway of DNA double-strand break repair. We show that RECQ1 interacts directly with the Ku70/80 subunit of the DNA-PK complex, and depletion of RECQ1 results in reduced end-joining in cell free extracts. In vitro, RECQ1 binds and unwinds the Ku70/80-bound partial duplex DNA substrate efficiently. Linear DNA is co-bound by RECQ1 and Ku70/80, and DNA binding by Ku70/80 is modulated by RECQ1. Collectively, these results provide the first evidence for an interaction of RECQ1 with Ku70/80 and a role of the human RecQ helicase in double-strand break repair through nonhomologous end-joining.
- Published
- 2013
30. Identification and Biochemical Characterization of a Novel Mutation in DDX11 Causing Warsaw Breakage Syndrome
- Author
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Mark E. Samuels, Robert M. Brosh, Jacques L. Michaud, Eliane Chouery, Sanjay Kumar Bharti, Guy A. Rouleau, Zoha Kibar, Fadi F. Hamdan, Joshua A. Sommers, Tony Yammine, André Mégarbané, Lysanne Patry, and Jose-Mario Capo-Chichi
- Subjects
Male ,Microcephaly ,Mutation, Missense ,Biology ,medicine.disease_cause ,Article ,DEAD-box RNA Helicases ,Consanguinity ,DDX11 ,Intellectual Disability ,Genetics ,medicine ,Missense mutation ,Humans ,Abnormalities, Multiple ,Exome ,Genetic Predisposition to Disease ,Amino Acid Sequence ,Genetics (clinical) ,Exome sequencing ,Family Health ,Mutation ,Base Sequence ,DNA Helicases ,Chromosome Breakage ,Sequence Analysis, DNA ,Syndrome ,medicine.disease ,Disease gene identification ,Molecular biology ,Pedigree ,Female ,Chromosome breakage - Abstract
Mutations in the gene encoding the iron–sulfur-containing DNA helicase DDX11 (ChlR1) were recently identified as a cause of a new recessive cohesinopathy, Warsaw breakage syndrome (WABS), in a single patient with severe microcephaly, pre- and postnatal growth retardation, and abnormal skin pigmentation. Here, using homozygosity mapping in a Lebanese consanguineous family followed by exome sequencing, we identified a novel homozygous mutation (c.788G>A [p.R263Q]) in DDX11 in three affected siblings with severe intellectual disability and many of the congenital abnormalities reported in the WABS original case. Cultured lymphocytes from the patients showed increased mitomycin C-induced chromosomal breakage, as found in WABS. Biochemical studies of purified recombinant DDX11 indicated that the p.R263Q mutation impaired DDX11 helicase activity by perturbing its DNA binding and DNA-dependent ATP hydrolysis. Our findings thus confirm the involvement of DDX11 in WABS, describe its phenotypical spectrum, and provide novel insight into the structural requirement for DDX11 activity. Hum Mutat 34:103-107, 2013.
- Published
- 2012
31. The Q motif of Fanconi anemia group J protein (FANCJ) DNA helicase regulates its dimerization, DNA binding, and DNA repair function
- Author
-
Caroline Kisker, Jason A. Loiland, Jochen Kuper, Hiroyuki Kitao, Yuliang Wu, Joshua A. Sommers, and Robert M. Brosh
- Subjects
Genome instability ,Glycerol ,Protein Denaturation ,DNA Repair ,DNA repair ,Cell Survival ,Amino Acid Motifs ,DNA and Chromosomes ,Biochemistry ,Models, Biological ,Catalysis ,law.invention ,Cell Line ,chemistry.chemical_compound ,Adenosine Triphosphate ,law ,Animals ,Humans ,Molecular Biology ,Oxazoles ,Adenosine Triphosphatases ,Chromatography ,biology ,Models, Genetic ,Hydrolysis ,DNA replication ,Temperature ,Helicase ,RNA ,Cell Biology ,RNA Helicase A ,Molecular biology ,Fanconi Anemia Complementation Group Proteins ,Recombinant Proteins ,Kinetics ,Basic-Leucine Zipper Transcription Factors ,chemistry ,Mutagenesis ,Mutation ,biology.protein ,Recombinant DNA ,Mutagenesis, Site-Directed ,Chickens ,Dimerization ,DNA ,HeLa Cells - Abstract
The Q motif, conserved in a number of RNA and DNA helicases, is proposed to be important for ATP binding based on structural data, but its precise biochemical functions are less certain. FANCJ encodes a Q motif DEAH box DNA helicase implicated in Fanconi anemia and breast cancer. A Q25A mutation of the invariant glutamine in the Q motif abolished its ability to complement cisplatin or telomestatin sensitivity of a fancj null cell line and exerted a dominant negative effect. Biochemical characterization of the purified recombinant FANCJ-Q25A protein showed that the mutation disabled FANCJ helicase activity and the ability to disrupt protein-DNA interactions. FANCJ-Q25A showed impaired DNA binding and ATPase activity but displayed ATP binding and temperature-induced unfolding transition similar to FANCJ-WT. Size exclusion chromatography and sedimentation velocity analyses revealed that FANCJ-WT existed as molecular weight species corresponding to a monomer and a dimer, and the dimeric form displayed a higher specific activity for ATPase and helicase, as well as greater DNA binding. In contrast, FANCJ-Q25A existed only as a monomer, devoid of helicase activity. Thus, the Q motif is essential for FANCJ enzymatic activity in vitro and DNA repair function in vivo.
- Published
- 2012
32. Physical and Functional Interaction Between Fanconi Anemia Group J Helicase and MRE11 Nuclease
- Author
-
Robert M. Brosh, Jean-Yves Masson, Avvaru N. Suhasini, Parameswary A. Muniandy, Yan Coulombe, Joshua A. Sommers, and Michael A. Seidman
- Subjects
Nuclease ,biology ,Chemistry ,Fanconi anemia ,Genetics ,biology.protein ,medicine ,Helicase ,medicine.disease ,Molecular Biology ,Biochemistry ,Molecular biology ,Biotechnology - Published
- 2012
33. Biochemical Characterization of Warsaw Breakage Syndrome Helicase
- Author
-
Joshua A. Sommers, Johan P. de Winter, Yuliang Wu, Robert M. Brosh, Irfan Khan, Human genetics, and CCA - Oncogenesis
- Subjects
DNA repair ,DNA polymerase ,DNA and Chromosomes ,Biochemistry ,Substrate Specificity ,DEAD-box RNA Helicases ,Control of chromosome duplication ,Humans ,Amino Acid Sequence ,Molecular Biology ,Sequence Deletion ,biology ,Circular bacterial chromosome ,DNA replication ,DNA Helicases ,Helicase ,Cell Biology ,DNA ,Syndrome ,RNA Helicase A ,DNA Repair-Deficiency Disorders ,Fanconi Anemia Complementation Group Proteins ,Basic-Leucine Zipper Transcription Factors ,biology.protein ,Primase - Abstract
Mutations in the human ChlR1 gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in sister chromatid cohesion and hypersensitivity to agents that induce replication stress. A role of ChlR1 helicase in sister chromatid cohesion was first evidenced by studies of the yeast homolog Chl1p; however, its cellular functions in DNA metabolism are not well understood. We carefully examined the DNA substrate specificity of purified recombinant human ChlR1 protein and the biochemical effect of a patient-derived mutation, a deletion of a single lysine (K897del) in the extreme C terminus of ChlR1. The K897del clinical mutation abrogated ChlR1 helicase activity on forked duplex or D-loop DNA substrates by perturbing its DNA binding and DNA-dependent ATPase activity. Wild-type ChlR1 required a minimal 5′ single-stranded DNA tail of 15 nucleotides to efficiently unwind a simple duplex DNA substrate. The additional presence of a 3′ single-stranded DNA tail as short as five nucleotides dramatically increased ChlR1 helicase activity, demonstrating the preference of the enzyme for forked duplex structures. ChlR1 unwound G-quadruplex (G4) DNA with a strong preference for a two-stranded antiparallel G4 (G2′) substrate and was only marginally active on a four-stranded parallel G4 structure. The marked difference in ChlR1 helicase activity on the G4 substrates, reflected by increased binding to the G2′ substrate, distinguishes ChlR1 from the sequence-related FANCJ helicase mutated in Fanconi anemia. The biochemical results are discussed in light of the known cellular defects associated with ChlR1 deficiency.
- Published
- 2012
34. Fanconi Anemia Group J Mutation Abolishes its DNA Repair Function by Uncoupling DNA Translocation from Helicase Activity
- Author
-
Joshua A. Sommers, Julianna S. Deakyne, Robert M. Brosh, Yuliang Wu, Thomas A. Leonard, Alexander V. Mazin, Kazuo Shin-ya, Hiroyuki Kitao, and Avvaru N. Suhasini
- Subjects
Mutation ,Fanconi anemia, complementation group C ,DNA repair ,Chemistry ,medicine.disease ,Dna translocation ,medicine.disease_cause ,Biochemistry ,Molecular biology ,Fanconi anemia ,Genetics ,medicine ,Molecular Biology ,Helicase activity ,Function (biology) ,Biotechnology - Published
- 2010
35. Molecular analyses of DNA helicases involved in the replicational stress response
- Author
-
Monika Aggarwal, Joshua A. Sommers, Avvaru N. Suhasini, Robert M. Brosh, and Yuliang Wu
- Subjects
chemistry.chemical_classification ,DNA Replication ,Base Sequence ,DNA repair ,Molecular Sequence Data ,DNA replication ,DNA Helicases ,Helicase ,Biology ,medicine.disease ,General Biochemistry, Genetics and Molecular Biology ,Article ,Nucleic acid metabolism ,chemistry.chemical_compound ,Enzyme ,chemistry ,Biochemistry ,medicine ,biology.protein ,Humans ,Molecular Biology ,Function (biology) ,DNA ,Werner syndrome - Abstract
The importance of helicases in nucleic acid metabolism and human disease has raised the bar for understanding how these unique enzymes function to perform their biological roles at the molecular level. Here we will describe experimental procedures and strategies to investigate the functions of helicases. These functional assays have been used to study DNA helicases important for the maintenance of genomic stability and genetically linked to age-related diseases and cancer. We will focus on the description of fluorometric helicase assays, protein displacement assays, and methods to characterize helicase activity on alternate DNA structures (triplex and quadruplex) used by our laboratory. The procedures to study these helicase functions are described in step-by-step detail to enable researchers interested in nucleic acid metabolism and related fields to apply these techniques to their own research questions.
- Published
- 2010
36. FANCJ Helicase Uniquely Senses Oxidative Base Damage in Either Strand of Duplex DNA and Is Stimulated by Replication Protein A to Unwind the Damaged DNA Substrate in a Strand-specific Manner*
- Author
-
Aaron C. Mason, Robert M. Brosh, Marc S. Wold, R. Daniel Camerini-Otero, Joshua A. Sommers, Avvaru N. Suhasini, and Oleg N. Voloshin
- Subjects
Guanine ,Breast Neoplasms ,Oxidative phosphorylation ,medicine.disease_cause ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,DNA Adducts ,Fanconi anemia ,Replication Protein A ,medicine ,Humans ,Molecular Biology ,Replication protein A ,Escherichia coli ,dnaB helicase ,biology ,DNA Helicases ,Helicase ,Cell Biology ,DNA ,medicine.disease ,Fanconi Anemia Complementation Group Proteins ,Enzyme Activation ,Oxidative Stress ,Basic-Leucine Zipper Transcription Factors ,Fanconi Anemia ,chemistry ,DNA: Replication, Repair, Recombination, and Chromosome Dynamics ,biology.protein ,Female ,Carcinogenesis ,Thymine ,DNA Damage - Abstract
FANCJ mutations are genetically linked to the Fanconi anemia complementation group J and predispose individuals to breast cancer. Understanding the role of FANCJ in DNA metabolism and how FANCJ dysfunction leads to tumorigenesis requires mechanistic studies of FANCJ helicase and its protein partners. In this work, we have examined the ability of FANCJ to unwind DNA molecules with specific base damage that can be mutagenic or lethal. FANCJ was inhibited by a single thymine glycol, but not 8-oxoguanine, in either the translocating or nontranslocating strands of the helicase substrate. In contrast, the human RecQ helicases (BLM, RECQ1, and WRN) display strand-specific inhibition of unwinding by the thymine glycol damage, whereas other DNA helicases (DinG, DnaB, and UvrD) are not significantly inhibited by thymine glycol in either strand. In the presence of replication protein A (RPA), but not Escherichia coli single-stranded DNA-binding protein, FANCJ efficiently unwound the DNA substrate harboring the thymine glycol damage in the nontranslocating strand; however, inhibition of FANCJ helicase activity by the translocating strand thymine glycol was not relieved. Strand-specific stimulation of human RECQ1 helicase activity was also observed, and RPA bound with high affinity to single-stranded DNA containing a single thymine glycol. Based on the biochemical studies, we propose a model for the specific functional interaction between RPA and FANCJ on the thymine glycol substrates. These studies are relevant to the roles of RPA, FANCJ, and other DNA helicases in the metabolism of damaged DNA that can interfere with basic cellular processes of DNA metabolism.
- Published
- 2009
37. FANCJ Uses Its Motor ATPase to Destabilize Protein-DNA Complexes, Unwind Triplexes, and Inhibit RAD51 Strand Exchange*
- Author
-
Alexander V. Mazin, Dmitry V. Bugreev, Sharon B. Cantor, Rigu Gupta, Robert M. Brosh, Joshua A. Sommers, and Nina A Rawtani
- Subjects
DNA Replication ,DNA Repair ,DNA repair ,RAD51 ,Biochemistry ,chemistry.chemical_compound ,Adenosine Triphosphate ,Humans ,DNA Breaks, Double-Stranded ,Molecular Biology ,Adenosine Triphosphatases ,Recombination, Genetic ,biology ,Hydrolysis ,DNA replication ,Helicase ,Cell Biology ,DNA ,Double Strand Break Repair ,Fanconi Anemia Complementation Group Proteins ,Homologous Recombination Pathway ,Basic-Leucine Zipper Transcription Factors ,chemistry ,DNA: Replication, Repair, Recombination, and Chromosome Dynamics ,biology.protein ,Female ,Rad51 Recombinase ,Homologous recombination - Abstract
Mutations in the FANCJ helicase predispose individuals to breast cancer and are genetically linked to the Fanconi anemia (FA) complementation group J. FA is a chromosomal instability disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high cancer risk. FANCJ has been proposed to function downstream of FANCD2 monoubiquitination, a critical event in the FA pathway. Evidence supports a role for FANCJ in a homologous recombination pathway of double strand break repair. In an effort to understand the molecular functions of FANCJ, we have investigated the ability of purified FANCJ recombinant protein to use its motor ATPase function for activities in addition to unwinding of conventional duplex DNA substrates. These efforts have led to the discovery that FANCJ ATP hydrolysis can be used to destabilize protein-DNA complexes and unwind triple helix alternate DNA structures. These novel catalytic functions of FANCJ may be important for its role in cellular DNA repair, recombination, or resolving DNA structural obstacles to replication. Consistent with this, we show that FANCJ can inhibit RAD51 strand exchange, an activity that is likely to be important for its role in controlling DNA repair through homologous recombination.
- Published
- 2009
38. Physical and functional mapping of the replication protein a interaction domain of the werner and bloom syndrome helicases
- Author
-
Kevin M. Doherty, Matthew D. Gray, Mark K. Kenny, Cayetano von Kobbe, Jae Wan Lee, Robert M. Brosh, Raichal P. Kureekattil, Nicolas H. Thomä, and Joshua A. Sommers
- Subjects
DNA Replication ,congenital, hereditary, and neonatal diseases and abnormalities ,Werner Syndrome Helicase ,Xenopus Proteins ,complex mixtures ,Biochemistry ,law.invention ,chemistry.chemical_compound ,law ,Replication Protein A ,Two-Hybrid System Techniques ,medicine ,Humans ,Bloom syndrome ,Binding site ,education ,Molecular Biology ,Replication protein A ,Adenosine Triphosphatases ,education.field_of_study ,Binding Sites ,biology ,RecQ Helicases ,DNA replication ,DNA Helicases ,nutritional and metabolic diseases ,Helicase ,Cell Biology ,DNA ,medicine.disease ,Molecular biology ,Cell biology ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,Exodeoxyribonucleases ,chemistry ,Recombinant DNA ,biology.protein - Abstract
The single-stranded DNA-binding protein replication protein A (RPA) interacts with several human RecQ DNA helicases that have important roles in maintaining genomic stability; however, the mechanism for RPA stimulation of DNA unwinding is not well understood. To map regions of Werner syndrome helicase (WRN) that interact with RPA, yeast two-hybrid studies, WRN affinity pull-down experiments and enzyme-linked immunosorbent assays with purified recombinant WRN protein fragments were performed. The results indicated that WRN has two RPA binding sites, a high affinity N-terminal site, and a lower affinity C-terminal site. Based on results from mapping studies, we sought to determine if the WRN N-terminal region harboring the high affinity RPA interaction site was important for RPA stimulation of WRN helicase activity. To accomplish this, we tested a catalytically active WRN helicase domain fragment (WRN(H-R)) that lacked the N-terminal RPA interaction site for its ability to unwind long DNA duplex substrates, which the wild-type enzyme can efficiently unwind only in the presence of RPA. WRN(H-R) helicase activity was significantly reduced on RPA-dependent partial duplex substrates compared with full-length WRN despite the presence of RPA. These results clearly demonstrate that, although WRN(H-R) had comparable helicase activity to full-length WRN on short duplex substrates, its ability to unwind RPA-dependent WRN helicase substrates was significantly impaired. Similarly, a Bloom syndrome helicase (BLM) domain fragment, BLM(642-1290), that lacked its N-terminal RPA interaction site also unwound short DNA duplex substrates similar to wild-type BLM, but was severely compromised in its ability to unwind long DNA substrates that full-length BLM helicase could unwind in the presence of RPA. These results suggest that the physical interaction between RPA and WRN or BLM helicases plays an important role in the mechanism for RPA stimulation of helicase-catalyzed DNA unwinding.
- Published
- 2005
39. Biochemical analysis of the DNA unwinding and strand annealing activities catalyzed by human RECQ1
- Author
-
Sheng Cui, Alessandro Vindigni, Joshua A. Sommers, Robert M. Brosh, Laura Muzzolini, Saba Choudhary, Jinnifer Korin Faulkner, Lucia Andreoli, and Sudha Sharma
- Subjects
Premature aging ,congenital, hereditary, and neonatal diseases and abnormalities ,Time Factors ,DNA repair ,Protein Conformation ,DNA, Single-Stranded ,Biochemistry ,Catalysis ,Substrate Specificity ,chemistry.chemical_compound ,Adenosine Triphosphate ,Annealing activity ,medicine ,Escherichia coli ,Humans ,Bloom syndrome ,Magnesium ,Molecular Biology ,Werner syndrome ,Adenosine Triphosphatases ,Ions ,Recombination, Genetic ,biology ,Dose-Response Relationship, Drug ,RecQ Helicases ,Circular bacterial chromosome ,DNA Helicases ,nutritional and metabolic diseases ,Helicase ,Cell Biology ,DNA ,medicine.disease ,Recombinant Proteins ,Protein Structure, Tertiary ,Kinetics ,chemistry ,biology.protein ,Nucleic Acid Conformation ,Electrophoresis, Polyacrylamide Gel ,Protein Binding - Abstract
RecQ helicases play an important role in preserving genomic integrity, and their cellular roles in DNA repair, recombination, and replication have been of considerable interest. Of the five human RecQ helicases identified, three are associated with genetic disorders characterized by an elevated incidence of cancer or premature aging: Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome. Although the biochemical properties and protein interactions of the WRN and BLM helicases defective in Werner syndrome and Bloom syndrome, respectively, have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. In this work, we have characterized the DNA substrate specificity and optimal cofactor requirements for efficient RECQ1-catalyzed DNA unwinding and determined that RECQ1 has certain properties that are distinct from those of other RecQ helicases. RECQ1 stably bound to a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. In addition to its DNA binding and helicase activities, RECQ1 catalyzed efficient strand annealing between complementary single-stranded DNA molecules. The ability of RECQ1 to promote strand annealing was modulated by ATP binding, which induced a conformational change in the protein. The enzymatic properties of the RECQ1 helicase and strand annealing activities are discussed in the context of proposed cellular DNA metabolic pathways that are important in the maintenance of genomic stability.
- Published
- 2005
40. Analysis of the DNA substrate specificity of the human BACH1 helicase associated with breast cancer
- Author
-
Sharon B. Cantor, Rigu Gupta, Robert M. Brosh, Sudha Sharma, Zhe Jin, and Joshua A. Sommers
- Subjects
DNA polymerase ,DNA, Single-Stranded ,Breast Neoplasms ,Biochemistry ,Substrate Specificity ,Humans ,Molecular Biology ,Replication protein A ,DNA, Cruciform ,DNA clamp ,biology ,Circular bacterial chromosome ,DNA Helicases ,Helicase ,Cell Biology ,DNA ,RNA Helicase A ,Branch migration ,Fanconi Anemia Complementation Group Proteins ,Enzyme Activation ,enzymes and coenzymes (carbohydrates) ,Basic-Leucine Zipper Transcription Factors ,biology.protein ,Biophysics ,Nucleic Acid Conformation ,Primase ,Transcription Factors - Abstract
We have investigated the DNA substrate specificity of BACH1 (BRCA1-associated C-terminal helicase). The importance of various DNA structural elements for efficient unwinding by purified recombinant BACH1 helicase was examined. The results indicated that BACH1 preferentially binds and unwinds a forked duplex substrate compared with a duplex flanked by only one single-stranded DNA (ssDNA) tail. In support of its DNA substrate preference, helicase sequestration studies revealed that BACH1 can be preferentially trapped by forked duplex molecules. BACH1 helicase requires a minimal 5 ' ssDNA tail of 15 nucleotides for unwinding of conventional duplex DNA substrates; however, the enzyme is able to catalytically release the third strand of the homologous recombination intermediate D-loop structure irrespective of DNA tail status. In contrast, BACH1 completely fails to unwind a synthetic Holliday junction structure. Moreover, BACH1 requires nucleic acid continuity in the 5 ' ssDNA tail of the forked duplex substrate within six nucleotides of the ssDNA-dsDNA junction to initiate efficiently DNA unwinding. These studies provide the first detailed information on the DNA substrate specificity of BACH1 helicase and provide insight to the types of DNA structures the enzyme is likely to act upon to perform its functions in DNA repair or recombination.
- Published
- 2005
41. In vivo function of the conserved non-catalytic domain of Werner syndrome helicase in DNA replication
- Author
-
Joshua A. Sommers, Sudha Sharma, and Robert M. Brosh
- Subjects
Genome instability ,DNA Replication ,congenital, hereditary, and neonatal diseases and abnormalities ,Saccharomyces cerevisiae Proteins ,Werner Syndrome Helicase ,Flap Endonucleases ,RecQ helicase ,Electrophoretic Mobility Shift Assay ,Saccharomyces cerevisiae ,chemistry.chemical_compound ,Control of chromosome duplication ,Catalytic Domain ,Genetics ,Humans ,Hydroxyurea ,Immunoprecipitation ,education ,Molecular Biology ,Genetics (clinical) ,Cells, Cultured ,Conserved Sequence ,Adenosine Triphosphatases ,education.field_of_study ,biology ,Okazaki fragments ,RecQ Helicases ,Cell Cycle ,Genetic Complementation Test ,DNA replication ,DNA Helicases ,nutritional and metabolic diseases ,Helicase ,General Medicine ,Protein Structure, Tertiary ,Exodeoxyribonucleases ,chemistry ,biology.protein ,Werner Syndrome ,DNA ,Protein Binding - Abstract
Werner syndrome is a genetic disorder characterized by genomic instability, elevated recombination and replication defects. The WRN gene encodes a RecQ helicase whose function(s) in cellular DNA metabolism is not well understood. To investigate the role of WRN in replication, we examined its ability to rescue cellular phenotypes of a yeast dna2 mutant defective in a helicase-endonuclease that participates with flap endonuclease 1 (FEN-1) in Okazaki fragment processing. Genetic complementation studies indicate that human WRN rescues dna2-1 mutant phenotypes of growth, cell cycle arrest and sensitivity to the replication inhibitor hydroxyurea or DNA damaging agent methylmethane sulfonate. A conserved non-catalytic C-terminal domain of WRN was sufficient for genetic rescue of dna2-1 mutant phenotypes. WRN and yeast FEN-1 were reciprocally co-immunoprecipitated from extracts of transformed dna2-1 cells. A physical interaction between yeast FEN-1 and WRN is demonstrated by yeast FEN-1 affinity pull-down experiments using transformed dna2-1 cells extracts and by ELISA assays with purified recombinant proteins. Biochemical analyses demonstrate that the C-terminal domain of WRN or BLM stimulates FEN-1 cleavage of its proposed physiological substrates during replication. Collectively, the results suggest that the WRN-FEN-1 interaction is biologically important in DNA metabolism and are consistent with a role of the conserved non-catalytic domain of a human RecQ helicase in DNA replication intermediate processing.
- Published
- 2004
42. WRN helicase and FEN-1 form a complex upon replication arrest and together process branchmigrating DNA structures associated with the replication fork
- Author
-
Sudha Sharma, Hui-I Kao, Henry C. Driscoll, Robert M. Brosh, Marit Otterlei, Robert A. Bambara, Grigory L. Dianov, and Joshua A. Sommers
- Subjects
DNA Replication ,congenital, hereditary, and neonatal diseases and abnormalities ,Werner Syndrome Helicase ,Flap Endonucleases ,Electrophoretic Mobility Shift Assay ,Biology ,Pre-replication complex ,DNA replication factor CDT1 ,Replication factor C ,Control of chromosome duplication ,Minichromosome maintenance ,Fluorescence Resonance Energy Transfer ,Humans ,education ,Molecular Biology ,S phase ,Genetics ,education.field_of_study ,RecQ Helicases ,DNA Helicases ,nutritional and metabolic diseases ,Articles ,Cell Biology ,Recombinant Proteins ,Cell biology ,Exodeoxyribonucleases ,biology.protein ,Origin recognition complex ,HeLa Cells ,Protein Binding - Abstract
Werner Syndrome is a premature aging disorder characterized by genomic instability, elevated recombination, and replication defects. It has been hypothesized that defective processing of certain replication fork structures by WRN may contribute to genomic instability. Fluorescence resonance energy transfer (FRET) analyses show that WRN and Flap Endonuclease-1 (FEN-1) form a complex in vivo that colocalizes in foci associated with arrested replication forks. WRN effectively stimulates FEN-1 cleavage of branch-migrating double-flap structures that are the physiological substrates of FEN-1 during replication. Biochemical analyses demonstrate that WRN helicase unwinds the chicken-foot HJ intermediate associated with a regressed replication fork and stimulates FEN-1 to cleave the unwound product in a structure-dependent manner. These results provide evidence for an interaction between WRN and FEN-1 in vivo and suggest that these proteins function together to process DNA structures associated with the replication fork.
- Published
- 2004
43. p53 Modulates the exonuclease activity of Werner syndrome protein
- Author
-
Joshua A. Sommers, Xin Wei Wang, Qin Yang, Elisa A. Spillare, Vilhelm A. Bohr, Robert M. Brosh, Curtis C. Harris, and Parimal Karmakar
- Subjects
Premature aging ,Genome instability ,Exonuclease ,congenital, hereditary, and neonatal diseases and abnormalities ,Werner Syndrome Helicase ,Nucleolus ,Mutant ,Molecular Sequence Data ,Apoptosis ,Biochemistry ,Catalysis ,medicine ,Humans ,Molecular Biology ,Werner syndrome ,Adenosine Triphosphatases ,Nucleoplasm ,biology ,Base Sequence ,RecQ Helicases ,DNA Helicases ,nutritional and metabolic diseases ,Helicase ,Cell Biology ,medicine.disease ,Molecular biology ,Exodeoxyribonucleases ,biology.protein ,Werner Syndrome ,Tumor Suppressor Protein p53 ,DNA Damage - Abstract
Werner syndrome (WS) is characterized by the early onset of symptoms of premature aging, cancer, and genomic instability. The molecular basis of the defects is not understood but presumably relates to the DNA helicase and exonuclease activities of the protein encoded by the WRN gene that is mutated in the disease. The attenuation of p53-mediated apoptosis in WS cells and reported physical interaction between WRN and the tumor suppressor p53 suggest that p53 and WRN functionally interact in a pathway necessary for the normal cellular response. In this study, we have demonstrated that p53 inhibits the exonuclease activity of the purified full-length recombinant WRN protein. p53 did not have an effect on a truncated amino-terminal WRN fragment that retains exonuclease activity but lacks the physical interaction domain for p53 located in the carboxyl terminus. Two naturally occurring p53 mutants found in human cancer displayed a reduced ability to inhibit WRN exonuclease activity. In cells arrested in S phase with hydroxyurea, WRN exits the nucleolus and colocalizes with p53 in the nucleoplasm. The regulation of WRN function by p53 is likely to play an important role in the maintenance of genomic integrity and prevention of cancer and other clinical symptoms associated with WS.
- Published
- 2001
44. Werner syndrome protein: biochemical properties and functional interactions
- Author
-
Amrita Machwe, Parimal Karmakar, Joshua A. Sommers, Robert M. Brosh, Vilhelm A. Bohr, David K. Orren, Jason Piotrowski, and Marcus P. Cooper
- Subjects
Exonuclease ,congenital, hereditary, and neonatal diseases and abnormalities ,Aging ,Werner Syndrome Helicase ,DNA Repair ,DNA damage ,DNA repair ,medicine.disease_cause ,Biochemistry ,Endocrinology ,Genetics ,medicine ,Humans ,education ,Molecular Biology ,Replication protein A ,Ku Autoantigen ,Werner syndrome ,education.field_of_study ,Mutation ,biology ,RecQ Helicases ,DNA Helicases ,nutritional and metabolic diseases ,Helicase ,Nuclear Proteins ,Antigens, Nuclear ,Cell Biology ,medicine.disease ,Molecular biology ,Cell biology ,DNA-Binding Proteins ,Exodeoxyribonucleases ,biology.protein ,Werner Syndrome ,DNA Damage - Abstract
Werner syndrome is a premature aging syndrome displaying numerous signs and symptoms found in normal aging. The disease is associated with a mutation in the WRN gene. We have purified the Werner protein (WRN) and studied its biochemical activities and its protein interactions. WRN is a helicase and an exonuclease and also has an associated ATPase activity. WRN interacts physically and functionally with replication protein A (RPA), which stimulates its helicase activity. We have studied the WRN exonuclease activity and found that it can be blocked by certain DNA lesions and not by others. Thus, while WRN does not bind to DNA damage, it may have properties that allow it to sense the presence of damage in DNA. More recently we have found other protein interactions that involve physical and functional interactions with WRN, which could suggest a role for WRN in DNA repair.
- Published
- 2000
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