99 results on '"Robert M. Brosh"'
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2. Special Issue: DNA Helicases: Mechanisms, Biological Pathways, and Disease Relevance
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Robert M. Brosh
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helicase ,replication ,DNA repair ,recombination ,transcription ,DNA structure ,Genetics ,QH426-470 - Abstract
DNA helicases have emerged as a prominent class of nucleic acid-metabolizing enzymes that play important roles in genome maintenance and cellular homeostasis [...]
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- 2021
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3. History of DNA Helicases
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Robert M. Brosh and Steven W. Matson
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helicase ,dna replication ,dna repair ,recombination ,transcription ,nucleic acid metabolism ,molecular biology ,human disease ,genomic instability ,science education ,Genetics ,QH426-470 - Abstract
Since the discovery of the DNA double helix, there has been a fascination in understanding the molecular mechanisms and cellular processes that account for: (i) the transmission of genetic information from one generation to the next and (ii) the remarkable stability of the genome. Nucleic acid biologists have endeavored to unravel the mysteries of DNA not only to understand the processes of DNA replication, repair, recombination, and transcription but to also characterize the underlying basis of genetic diseases characterized by chromosomal instability. Perhaps unexpectedly at first, DNA helicases have arisen as a key class of enzymes to study in this latter capacity. From the first discovery of ATP-dependent DNA unwinding enzymes in the mid 1970’s to the burgeoning of helicase-dependent pathways found to be prevalent in all kingdoms of life, the story of scientific discovery in helicase research is rich and informative. Over four decades after their discovery, we take this opportunity to provide a history of DNA helicases. No doubt, many chapters are left to be written. Nonetheless, at this juncture we are privileged to share our perspective on the DNA helicase field − where it has been, its current state, and where it is headed.
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- 2020
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4. New Insights Into DNA Helicases as Druggable Targets for Cancer Therapy
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Arindam Datta and Robert M. Brosh
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helicase ,DNA repair ,replication ,genomic instability ,small molecule ,therapy ,Biology (General) ,QH301-705.5 - Abstract
Small molecules that deter the functions of DNA damage response machinery are postulated to be useful for enhancing the DNA damaging effects of chemotherapy or ionizing radiation treatments to combat cancer by impairing the proliferative capacity of rapidly dividing cells that accumulate replicative lesions. Chemically induced or genetic synthetic lethality is a promising area in personalized medicine, but it remains to be optimized. A new target in cancer therapy is DNA unwinding enzymes known as helicases. Helicases play critical roles in all aspects of nucleic acid metabolism. We and others have investigated small molecule targeted inhibition of helicase function by compound screens using biochemical and cell-based approaches. Small molecule-induced trapping of DNA helicases may represent a generalized mechanism exemplified by certain topoisomerase and PARP inhibitors that exert poisonous consequences, especially in rapidly dividing cancer cells. Taking the lead from the broader field of DNA repair inhibitors and new information gleaned from structural and biochemical studies of DNA helicases, we predict that an emerging strategy to identify useful helicase-interacting compounds will be structure-based molecular docking interfaced with a computational approach. Potency, specificity, drug resistance, and bioavailability of helicase inhibitor drugs and targeting such compounds to subcellular compartments where the respective helicases operate must be addressed. Beyond cancer therapy, continued and new developments in this area may lead to the discovery of helicase-interacting compounds that chemically rescue clinically relevant helicase missense mutant proteins or activate the catalytic function of wild-type DNA helicases, which may have novel therapeutic application.
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- 2018
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5. Holding All the Cards—How Fanconi Anemia Proteins Deal with Replication Stress and Preserve Genomic Stability
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Arindam Datta and Robert M. Brosh
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Fanconi anemia ,genomic instability ,DNA repair ,DNA replication ,genetic diseases ,cancer ,chromosome ,helicase ,Genetics ,QH426-470 - Abstract
Fanconi anemia (FA) is a hereditary chromosomal instability disorder often displaying congenital abnormalities and characterized by a predisposition to progressive bone marrow failure (BMF) and cancer. Over the last 25 years since the discovery of the first linkage of genetic mutations to FA, its molecular genetic landscape has expanded tremendously as it became apparent that FA is a disease characterized by a defect in a specific DNA repair pathway responsible for the correction of covalent cross-links between the two complementary strands of the DNA double helix. This pathway has become increasingly complex, with the discovery of now over 20 FA-linked genes implicated in interstrand cross-link (ICL) repair. Moreover, gene products known to be involved in double-strand break (DSB) repair, mismatch repair (MMR), and nucleotide excision repair (NER) play roles in the ICL response and repair of associated DNA damage. While ICL repair is predominantly coupled with DNA replication, it also can occur in non-replicating cells. DNA damage accumulation and hematopoietic stem cell failure are thought to contribute to the increased inflammation and oxidative stress prevalent in FA. Adding to its confounding nature, certain FA gene products are also engaged in the response to replication stress, caused endogenously or by agents other than ICL-inducing drugs. In this review, we discuss the mechanistic aspects of the FA pathway and the molecular defects leading to elevated replication stress believed to underlie the cellular phenotypes and clinical features of FA.
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- 2019
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6. Molecular and cellular functions of the FANCJ DNA helicase defective in cancer and in Fanconi Anemia
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Robert M. Brosh and Sharon B. Cantor
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DNA Repair ,Fanconi Anemia ,Cancer ,replication ,genomic stability ,tumor suppressor ,Genetics ,QH426-470 - Abstract
The FANCJ DNA helicase is mutated in hereditary breast and ovarian cancer as well as the progressive bone marrow failure disorder Fanconi anemia (FA). FANCJ is linked to cancer suppression and DNA double strand break (DSB) repair through its direct interaction with the hereditary breast cancer associated gene product, BRCA1. FANCJ also operates in the FA pathway of interstrand cross-link (ICL) repair and contributes to homologous recombination (HR). FANCJ collaborates with a number of DNA metabolizing proteins implicated in DNA damage detection and repair, and plays an important role in cell cycle checkpoint control. In addition to its role in the classical FA pathway, FANCJ is believed to have other functions that are centered on alleviating replication stress. FANCJ resolves G-quadruplex (G4) DNA structures that are known to affect cellular replication and transcription, and potentially plays a role in the preservation and functionality of chromosomal structures such as telomeres. Recent studies suggest that FANCJ helps to maintain chromatin structure and preserve epigenetic stability by facilitating smooth progression of the replication fork when it encounters DNA damage or an alternate DNA structure such as a G4. Ongoing studies suggest a prominent but still not well-understood role of FANCJ in transcriptional regulation, chromosomal structure and function, and DNA damage repair to maintain genomic stability. This review will synthesize our current understanding of the molecular and cellular functions of FANCJ that are critical for chromosomal integrity.
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- 2014
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7. FANCJ compensates for RAP80 deficiency and suppresses genomic instability induced by interstrand cross-links
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Robert M. Brosh, Arindam Datta, Marina A. Bellani, Sanket Awate, Christopher A. Dunn, Joshua A. Sommers, Michael M. Seidman, Sumeet Nayak, George Lucian Moldovan, Claudia M. Nicolae, Olivia Yang, and Sharon B. Cantor
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Genome instability ,DNA Repair ,DNA damage ,DNA repair ,Mitomycin ,RAD51 ,Genome Integrity, Repair and Replication ,Genomic Instability ,Gene Knockout Techniques ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Chromosomal Instability ,Genetics ,Humans ,BRIP1 Gene ,DNA Breaks, Double-Stranded ,Histone Chaperones ,030304 developmental biology ,0303 health sciences ,biology ,BRCA1 Protein ,Recombinational DNA Repair ,Helicase ,Fanconi Anemia Complementation Group Proteins ,Cell biology ,DNA-Binding Proteins ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,Rad51 Recombinase ,Homologous recombination ,RNA Helicases ,DNA ,DNA Damage ,HeLa Cells - Abstract
FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ’s catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.
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- 2020
8. DNA helicases and their roles in cancer
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Arindam Datta, Srijita Dhar, and Robert M. Brosh
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Genome instability ,DNA Replication ,DNA Repair ,DNA damage ,DNA repair ,Biochemistry ,Genomic Instability ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Neoplasms ,Animals ,Humans ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,DNA Helicases ,Helicase ,Cell Biology ,Telomere ,Cell biology ,Helicase Gene ,chemistry ,030220 oncology & carcinogenesis ,Cancer cell ,biology.protein ,DNA - Abstract
DNA helicases, known for their fundamentally important roles in genomic stability, are high profile players in cancer. Not only are there monogenic helicase disorders with a strong disposition to cancer, it is well appreciated that helicase variants are associated with specific cancers (e.g., breast cancer). Flipping the coin, DNA helicases are frequently overexpressed in cancerous tissues and reduction in helicase gene expression results in reduced proliferation and growth capacity, as well as DNA damage induction and apoptosis of cancer cells. The seminal roles of helicases in the DNA damage and replication stress responses, as well as DNA repair pathways, validate their vital importance in cancer biology and suggest their potential values as targets in anti-cancer therapy. In recent years, many laboratories have characterized the specialized roles of helicase to resolve transcription-replication conflicts, maintain telomeres, mediate cell cycle checkpoints, remodel stalled replication forks, and regulate transcription. In vivo models, particularly mice, have been used to interrogate helicase function and serve as a bridge for preclinical studies that may lead to novel therapeutic approaches. In this review, we will summarize our current knowledge of DNA helicases and their roles in cancer, emphasizing the latest developments.
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- 2020
9. 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.
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- 2020
10. Synthetic Lethal Interactions of RECQ Helicases
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Robert M. Brosh, Arindam Datta, Sanket Awate, and Srijita Dhar
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0301 basic medicine ,DNA Replication ,Cancer Research ,DNA Repair ,DNA repair ,Antineoplastic Agents ,Synthetic lethality ,Biology ,Medical Oncology ,Genome ,Genomic Instability ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Cell Line, Tumor ,Neoplasms ,medicine ,Animals ,Humans ,Precision Medicine ,Gene ,Rothmund–Thomson syndrome ,Werner syndrome ,Genetics ,RecQ Helicases ,Helicase ,medicine.disease ,Xenograft Model Antitumor Assays ,030104 developmental biology ,Oncology ,chemistry ,030220 oncology & carcinogenesis ,Gene Knockdown Techniques ,Mutation ,biology.protein ,Synthetic Lethal Mutations ,DNA - Abstract
DNA helicases have risen to the forefront as genome caretakers. Their prominent roles in chromosomal stability is demonstrated by the linkage of mutations in helicase genes to hereditary disorders with defects in DNA repair, the replication stress response, and/or transcriptional activation. Conversely, accumulating evidence suggests that in cancer cells DNA helicases have a network of pathway interactions such that co-deficiency of certain helicases and their genetically interacting proteins result in synthetic lethality (SL). Such genetic interactions may potentially be exploited for cancer therapies. We will discuss the roles of RECQ DNA helicases in cancer, emphasizing some of the more recent developments in synthetic lethality.
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- 2020
11. BLM’s balancing act and the involvement of FANCJ in DNA repair
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Srijita Dhar and Robert M. Brosh
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0301 basic medicine ,Genome instability ,congenital, hereditary, and neonatal diseases and abnormalities ,biology ,DNA damage ,DNA repair ,Mutagenesis ,nutritional and metabolic diseases ,Helicase ,Cell Biology ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,Chromosome instability ,biology.protein ,Molecular Biology ,Gene ,DNA ,Developmental Biology - Abstract
Timely recruitment of DNA damage response proteins to sites of genomic structural lesions is very important for signaling mechanisms to activate appropriate cell cycle checkpoints but also repair the altered DNA sequence to suppress mutagenesis. The eukaryotic cell is characterized by a complex cadre of players and pathways to ensure genomic stability in the face of replication stress or outright genomic insult by endogenous metabolites or environmental agents. Among the key performers are molecular motor DNA unwinding enzymes known as helicases that sense genomic perturbations and separate structured DNA strands so that replacement of a damaged base or sugar-phosphate backbone lesion can occur efficiently. Mutations in the BLM gene encoding the DNA helicase BLM leads to a rare chromosomal instability disorder known as Bloom's syndrome. In a recent paper by the Sengupta lab, BLM's role in the correction of double-strand breaks (DSB), a particularly dangerous form of DNA damage, was investigated. Adding to the complexity, BLM appears to be a key ringmaster of DSB repair as it acts both positively and negatively to regulate correction pathways of high or low fidelity. The FANCJ DNA helicase, mutated in another chromosomal instability disorder known as Fanconi Anemia, is an important player that likely coordinates with BLM in the balancing act. Further studies to dissect the roles of DNA helicases like FANCJ and BLM in DSB repair are warranted.
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- 2018
12. A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair
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Sanjay Kumar Bharti, Lynda Bradley, Robert M. Brosh, Joshua A. Sommers, Keir C. Neuman, Irfan Khan, Sanket Awate, Marina A. Bellani, Kazuo Shin-ya, Graeme A. King, Koji Kobayashi, Yuliang Wu, Dana Branzei, Marc S. Wold, Takuye Abe, Yeonee Seol, Hiroyuki Kitao, and Venkatasubramanian Vidhyasagar
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0301 basic medicine ,Aphidicolin ,DNA Replication ,DNA Repair ,DNA damage ,DNA repair ,Mutation, Missense ,DNA, Single-Stranded ,Cell Line ,Recombinases ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Stress, Physiological ,Replication Protein A ,Genetics ,Animals ,DNA Breaks, Double-Stranded ,Replication protein A ,Oxazoles ,Adenosine Triphosphatases ,biology ,Nucleic Acid Enzymes ,DNA replication ,DNA Helicases ,Helicase ,Processivity ,Fanconi Anemia Complementation Group Proteins ,Cell biology ,G-Quadruplexes ,030104 developmental biology ,Fanconi Anemia ,chemistry ,030220 oncology & carcinogenesis ,Checkpoint Kinase 1 ,biology.protein ,DNA Polymerase Inhibitor ,Rad51 Recombinase ,Cisplatin ,Chickens ,RNA Helicases - Abstract
Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj−/− cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj−/− cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.
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- 2018
13. Cockayne syndrome group A and B proteins converge on transcription-linked resolution of non-B DNA
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Paul Bastian, Supriyo De, Anne Tseng, Soumita Ghosh, Sanjay Kumar Bharti, Teruaki Iyama, Karsten Scheibye-Alsing, Evandro Fei Fang, Ilya G. Goldberg, Robert M. Brosh, Myriam Gorospe, Krisztina Marosi, Robert W. Maul, Henok Kassahun, Mark P. Mattson, Martin Borch Jensen, Lynn Froetscher, Morten Scheibye-Knudsen, Hilde Nilsen, Vilhelm A. Bohr, David Mark Eckley, and David M. Wilson
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0301 basic medicine ,DNA Repair ,Transcription, Genetic ,DNA damage ,Poly (ADP-Ribose) Polymerase-1 ,Biology ,DNA, Ribosomal ,Cockayne syndrome ,law.invention ,Neuroblastoma ,03 medical and health sciences ,PARP1 ,law ,Transcription (biology) ,Cell Line, Tumor ,medicine ,Humans ,Cockayne Syndrome ,Poly-ADP-Ribose Binding Proteins ,Gene ,Ribosomal DNA ,Polymerase ,Multidisciplinary ,DNA Helicases ,DNA, Neoplasm ,Biological Sciences ,medicine.disease ,Molecular biology ,G-Quadruplexes ,DNA Repair Enzymes ,030104 developmental biology ,Gene Knockdown Techniques ,Recombinant DNA ,biology.protein ,DNA Damage ,Transcription Factors - Abstract
Cockayne syndrome is a neurodegenerative accelerated aging disorder caused by mutations in the CSA or CSB genes. Although the pathogenesis of Cockayne syndrome has remained elusive, recent work implicates mitochondrial dysfunction in the disease progression. Here, we present evidence that loss of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number of cell lines. Furthermore, machine-learning algorithms predict that diseases with defects in ribosomal DNA (rDNA) transcription have mitochondrial dysfunction, and, accordingly, this is found when factors involved in rDNA transcription are knocked down. Mechanistically, loss of CSA or CSB leads to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans In conclusion, this work supports a role for impaired ribosomal DNA transcription in Cockayne syndrome and suggests that transcription-coupled resolution of secondary structures may be a mechanism to repress spurious activation of a DNA damage response.
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- 2016
14. Cellular Assays to Study the Functional Importance of Human DNA Repair Helicases
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Robert M. Brosh, Srijita Dhar, Joshua A. Sommers, and Sanket Awate
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0301 basic medicine ,DNA Repair ,DNA repair ,DNA damage ,Cellular homeostasis ,Apoptosis ,Cell Count ,Genomic Instability ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cell Line, Tumor ,Humans ,Cell Proliferation ,Enzyme Assays ,biology ,Chemistry ,DNA Helicases ,Helicase ,Cell biology ,genomic DNA ,030104 developmental biology ,Microscopy, Fluorescence ,Polynucleotide ,030220 oncology & carcinogenesis ,biology.protein ,Biological Assay ,Homologous recombination ,DNA - Abstract
DNA helicases represent a specialized class of enzymes that play crucial roles in the DNA damage response. Using the energy of nucleoside triphosphate binding and hydrolysis, helicases behave as molecular motors capable of efficiently disrupting the many noncovalent hydrogen bonds that stabilize DNA molecules with secondary structure. In addition to their importance in DNA damage sensing and signaling, DNA helicases facilitate specific steps in DNA repair mechanisms that require polynucleotide tract unwinding or resolution. Because they play fundamental roles in the DNA damage response and DNA repair, defects in helicases disrupt cellular homeostasis. Thus, helicase deficiency or inhibition may result in reduced cell proliferation and survival, apoptosis, DNA damage induction, defective localization of repair proteins to sites of genomic DNA damage, chromosomal instability, and defective DNA repair pathways such as homologous recombination of double-strand breaks. In this chapter, we will describe step-by-step protocols to assay the functional importance of human DNA repair helicases in genome stability and cellular homeostasis.
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- 2019
15. A high-throughput screen to identify novel small molecule inhibitors of the Werner Syndrome Helicase-Nuclease (WRN)
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Robert M. Brosh, Vilhelm A. Bohr, Thomas S. Dexheimer, Tomasz Kulikowicz, Joshua A. Sommers, Dorjbal Dorjsuren, Deborah L. Croteau, Anton Simeonov, David J. Maloney, and Ajit Jadhav
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0301 basic medicine ,Werner Syndrome Helicase ,DNA repair ,Science ,Synthetic lethality ,Small Molecule Libraries ,Inhibitory Concentration 50 ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cell Line, Tumor ,medicine ,Humans ,Fluorometry ,Enzyme Inhibitors ,education ,Cell Proliferation ,Enzyme Assays ,Werner syndrome ,education.field_of_study ,Multidisciplinary ,biology ,Chemistry ,DNA replication ,Reproducibility of Results ,Helicase ,DNA ,medicine.disease ,High-Throughput Screening Assays ,Cell biology ,030104 developmental biology ,030220 oncology & carcinogenesis ,Cancer cell ,Biocatalysis ,biology.protein ,Medicine - Abstract
Werner syndrome (WS), an autosomal recessive genetic disorder, displays accelerated clinical symptoms of aging leading to a mean lifespan less than 50 years. The WS helicase-nuclease (WRN) is involved in many important pathways including DNA replication, recombination and repair. Replicating cells are dependent on helicase activity, leading to the pursuit of human helicases as potential therapeutic targets for cancer treatment. Small molecule inhibitors of DNA helicases can be used to induce synthetic lethality, which attempts to target helicase-dependent compensatory DNA repair pathways in tumor cells that are already genetically deficient in a specific pathway of DNA repair. Alternatively, helicase inhibitors may be useful as tools to study the specialized roles of helicases in replication and DNA repair. In this study, approximately 350,000 small molecules were screened based on their ability to inhibit duplex DNA unwinding by a catalytically active WRN helicase domain fragment in a high-throughput fluorometric assay to discover new non-covalent small molecule inhibitors of the WRN helicase. Select compounds were screened to exclude ones that inhibited DNA unwinding by other helicases in the screen, bound non-specifically to DNA, acted as irreversible inhibitors, or possessed unfavorable chemical properties. Several compounds were tested for their ability to impair proliferation of cultured tumor cells. We observed that two of the newly identified WRN helicase inhibitors inhibited proliferation of cancer cells in a lineage-dependent manner. These studies represent the first high-throughput screen for WRN helicase inhibitors and the results have implications for anti-cancer strategies targeting WRN in different cancer cells and genetic backgrounds.
- Published
- 2019
16. 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.
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- 2016
17. 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
18. Helicases and Their Relevance to Aging
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Sanjay Kumar Bharti, Jack D. Crouch, Sanket Awate, Taraswi Banerjee, and Robert M. Brosh
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biology ,DNA repair ,Neurodegeneration ,Helicase ,Computational biology ,medicine.disease ,Nucleic acid metabolism ,chemistry.chemical_compound ,chemistry ,Chromosome instability ,biology.protein ,medicine ,Transcriptional regulation ,Gene ,DNA - Abstract
Molecular motor DNA unwinding enzymes known as helicases play prominent roles in cellular nucleic acid metabolism. A number of hereditary disorders with accelerated aging features have been identified in which the hereditary mutations reside in genes encoding DNA helicases. This has prompted tremendous interest in the aging community to characterize the molecular and cellular functions of these clinically relevant helicases and how their roles and pathways are important for the suppression of chromosomal instability, age-related diseases, neurodegeneration, and cancer. In this chapter, we focused our attention on some of the latest research developments pertaining to roles of helicases in DNA repair, transcriptional regulation, and genome maintenance, with a discussion of valuable mouse models. Progress in the field has provided new insight into the functional roles of helicases at the molecular, cellular, tissue-wide, and organismal levels, providing a launchpad for a more mechanistic understanding of age-related diseases and potential or emerging therapies.
- Published
- 2018
19. RecQ and Fe–S helicases have unique roles in DNA metabolism dictated by their unwinding directionality, substrate specificity, and protein interactions
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Robert M. Brosh and Katrina N Estep
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0301 basic medicine ,Iron-Sulfur Proteins ,DNA repair ,Biochemistry ,Article ,Nucleic acid metabolism ,Protein–protein interaction ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Neoplasms ,Molecular motor ,Humans ,Gene ,Regulation of gene expression ,Genetics ,Chromosome Aberrations ,biology ,RecQ Helicases ,DNA Helicases ,Helicase ,DNA ,030104 developmental biology ,chemistry ,Mutation ,biology.protein ,Protein Binding - Abstract
Helicases are molecular motors that play central roles in nucleic acid metabolism. Mutations in genes encoding DNA helicases of the RecQ and iron–sulfur (Fe–S) helicase families are linked to hereditary disorders characterized by chromosomal instabilities, highlighting the importance of these enzymes. Moreover, mono-allelic RecQ and Fe–S helicase mutations are associated with a broad spectrum of cancers. This review will discuss and contrast the specialized molecular functions and biological roles of RecQ and Fe–S helicases in DNA repair, the replication stress response, and the regulation of gene expression, laying a foundation for continued research in these important areas of study.
- Published
- 2017
20. Protein Degradation Pathways Regulate the Functions of Helicases in the DNA Damage Response and Maintenance of Genomic Stability
- Author
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Joshua A. Sommers, Robert M. Brosh, and Avvaru N. Suhasini
- Subjects
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
21. What is wrong with Fanconi anemia cells?
- Author
-
Sharon B. Cantor and Robert M. Brosh
- Subjects
DNA Repair ,DNA damage ,DNA repair ,Review ,Biology ,medicine.disease_cause ,Fanconi anemia ,medicine ,Humans ,Molecular Biology ,Genetics ,DNA replication ,Nuclear Proteins ,Cell Biology ,medicine.disease ,Fanconi Anemia Complementation Group Proteins ,DNA Repair Enzymes ,Fanconi Anemia ,MutS Homolog 2 Protein ,MSH2 ,Cancer cell ,Cancer research ,DNA mismatch repair ,Carcinogenesis ,DNA Damage ,Protein Binding ,Developmental Biology - Abstract
Figuring out what is wrong in Fanconi anemia (FA) patient cells is critical to understanding the contributions of the FA pathway to DNA repair and tumor suppression. Although FA patients exhibit a wide range of disease manifestation as well as severity (asymptomatic to congenital abnormalities, bone marrow failure, and cancer), cells from FA patients share underlying defects in their ability to process DNA lesions that interfere with DNA replication. In particular, FA cells are very sensitive to agents that induce DNA interstrand crosslinks (ICLs). The cause of this pronounced ICL sensitivity is not fully understood, but has been linked to the aberrant activation of DNA damage repair proteins, checkpoints and pathways. Thus, regulation of these responses through coordination of repair processing at stalled replication forks is an essential function of the FA pathway. Here, we briefly summarize some of the aberrant DNA damage responses contributing to defects in FA cells, and detail the newly-identified relationship between FA and the mismatch repair protein, MSH2. Understanding the contribution of MSH2 and/or other proteins to the replication problem in FA cells will be key to assessing therapeutic options to improve the health of FA patients. Moreover, loss of these factors, if linked to improved replication, could be a key event in the progression of FA cells to cancer cells. Likewise, loss of these factors could synergize to enhance tumorigenesis or confer chemoresistance in tumors defective in FA-BRCA pathway proteins and provide a basis for biomarkers for disease progression and response.
- Published
- 2014
22. Detection of G-quadruplex DNA in mammalian cells
- Author
-
Elizabeth A. Chavez, Jesse M. Platt, Yu Chuan Huang, F. Brad Johnson, Dipankar Sen, Peter M. Lansdorp, Robert M. Brosh, Alexander Harper Hewitt Henderson, Yuliang Wu, and Damage and Repair in Cancer Development and Cancer Treatment (DARE)
- Subjects
0301 basic medicine ,Expression of Concern ,medicine.drug_class ,DNA repair ,Biology ,Gene Regulation, Chromatin and Epigenetics ,010402 general chemistry ,Monoclonal antibody ,G-quadruplex ,SEQUENCE ,01 natural sciences ,Chromosomes ,SACCHAROMYCES-CEREVISIAE ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Genetics ,medicine ,Animals ,Humans ,CRYSTAL-STRUCTURE ,NUCLEIC-ACIDS ,OXYTRICHA TELOMERIC DNA ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,BINDING PROTEINS ,Helicase ,Antibodies, Monoclonal ,DNA ,Molecular biology ,Fanconi Anemia Complementation Group Proteins ,Cell biology ,0104 chemical sciences ,3. Good health ,Proliferating cell nuclear antigen ,G-Quadruplexes ,Cell nucleus ,030104 developmental biology ,medicine.anatomical_structure ,ANTIBODY ,chemistry ,Nucleic acid ,biology.protein ,COMPLEXES ,GENOMIC STABILITY ,G-QUARTET - Abstract
It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal antibodies specific for different G4 DNA structures. We show that one of these antibodies designated 1H6 exhibits strong nuclear staining in most human and murine cells. Staining intensity increased on treatment of cells with agents that stabilize G4 DNA and, strikingly, cells deficient in FANCJ, a G4 DNA-specific helicase, showed stronger nuclear staining than controls. Our data strongly support the existence of G4 DNA structures in mammalian cells and indicate that the abundance of such structures is increased in the absence of FANCJ. We conclude that monoclonal antibody 1H6 is a valuable tool for further studies on the role of G4 DNA in cell and molecular biology.
- Published
- 2017
23. Molecular functions and cellular roles of the ChlR1 (DDX11) helicase defective in the rare cohesinopathy Warsaw breakage syndrome
- Author
-
Robert M. Brosh, Sanjay Kumar Bharti, Taraswi Banerjee, Yuliang Wu, Joshua A. Sommers, and Irfan Khan
- Subjects
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
24. Editorial
- Author
-
Robert M. Brosh Tomeny
- Subjects
0301 basic medicine ,Gerontology ,Aging ,DNA Repair ,business.industry ,Aging, Premature ,Biochemistry ,Article ,03 medical and health sciences ,030104 developmental biology ,Neurology ,Ageing ,Medicine ,Humans ,business ,Molecular Biology ,Biotechnology ,DNA Damage - Published
- 2016
25. Werner Syndrome Helicase Has a Critical Role in DNA Damage Responses in the Absence of a Functional Fanconi Anemia Pathway
- Author
-
Monika Aggarwal, Chiara Iannascoli, Taraswi Banerjee, Pietro Pichierri, Joshua A. Sommers, Robert M. Brosh, and Robert H. Shoemaker
- Subjects
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
26. DNA helicases involved in DNA repair and their roles in cancer
- Author
-
Robert M. Brosh
- Subjects
Genome instability ,Genetics ,DNA Repair ,biology ,DNA damage ,DNA repair ,Applied Mathematics ,General Mathematics ,DNA Helicases ,Helicase ,Cellular homeostasis ,Eukaryotic DNA replication ,Computational biology ,RNA Helicase A ,Genomic Instability ,Article ,Neoplasms ,biology.protein ,Humans ,DNA mismatch repair - Abstract
Helicases have major roles in genome maintenance by unwinding structured nucleic acids. Their prominence is marked by various cancers and genetic disorders that are linked to helicase defects. Although considerable effort has been made to understand the functions of DNA helicases that are important for genomic stability and cellular homeostasis, the complexity of the DNA damage response leaves us with unanswered questions regarding how helicase-dependent DNA repair pathways are regulated and coordinated with cell cycle checkpoints. Further studies may open the door to targeting helicases in order to improve cancer treatments based on DNA-damaging chemotherapy or radiation.
- Published
- 2013
27. Fanconi Anemia Group J Helicase and MRE11 Nuclease Interact To Facilitate the DNA Damage Response
- Author
-
Jean-Yves Masson, Michael M. Seidman, Parameswary A. Muniandy, Robert M. Brosh, Joshua A. Sommers, Yan Coulombe, Avvaru N. Suhasini, and Sharon B. Cantor
- Subjects
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
28. Mechanistic and biological considerations of oxidatively damaged DNA for helicase-dependent pathways of nucleic acid metabolism
- Author
-
Robert M. Brosh and Jack D. Crouch
- Subjects
0301 basic medicine ,Aging ,DNA repair ,DNA damage ,Carcinogenesis ,Biology ,medicine.disease_cause ,Biochemistry ,Article ,Nucleic acid metabolism ,03 medical and health sciences ,chemistry.chemical_compound ,Transcription (biology) ,Physiology (medical) ,Chromosomal Instability ,Nucleic Acids ,medicine ,Animals ,Humans ,DNA Helicases ,Helicase ,DNA ,Oxidative Stress ,030104 developmental biology ,chemistry ,biology.protein ,Nucleoside triphosphate ,Reactive Oxygen Species ,Oxidation-Reduction ,Oxidative stress ,Signal Transduction - Abstract
Cells are under constant assault from reactive oxygen species that occur endogenously or arise from environmental agents. An important consequence of such stress is the generation of oxidatively damaged DNA, which is represented by a wide range of non-helix distorting and helix-distorting bulkier lesions that potentially affect a number of pathways including replication and transcription; consequently DNA damage tolerance and repair pathways are elicited to help cells cope with the lesions. The cellular consequences and metabolism of oxidatively damaged DNA can be quite complex with a number of DNA metabolic proteins and pathways involved. Many of the responses to oxidative stress involve a specialized class of enzymes known as helicases, the topic of this review. Helicases are molecular motors that convert the energy of nucleoside triphosphate hydrolysis to unwinding of structured polynucleic acids. Helicases by their very nature play fundamentally important roles in DNA metabolism and are implicated in processes that suppress chromosomal instability, genetic disease, cancer, and aging. We will discuss the roles of helicases in response to nuclear and mitochondrial oxidative stress and how this important class of enzymes help cells cope with oxidatively generated DNA damage through their functions in the replication stress response, DNA repair, and transcriptional regulation.
- Published
- 2016
29. CDK1 phosphorylates WRN at collapsed replication forks
- Author
-
Sara Rinalducci, Joshua A. Sommers, Robert M. Brosh, Francesca Grillini, Massimo Sanchez, Pietro Pichierri, Lello Zolla, Annapaola Franchitto, and Valentina Palermo
- Subjects
DNA Replication ,Genetics and Molecular Biology (all) ,0301 basic medicine ,Genome instability ,congenital, hereditary, and neonatal diseases and abnormalities ,Werner Syndrome Helicase ,DNA Repair ,DNA repair ,Science ,General Physics and Astronomy ,Biology ,environment and public health ,Biochemistry ,Article ,Genomic Instability ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,03 medical and health sciences ,CDC2 Protein Kinase ,Humans ,DNA Breaks, Double-Stranded ,DSBs repair pathway ,Phosphorylation ,Homologous Recombination ,education ,Regulation of gene expression ,Genetics ,Cyclin-dependent kinase 1 ,education.field_of_study ,Biochemistry, Genetics and Molecular Biology (all) ,DNA Repair, DSBs repair pathway ,Multidisciplinary ,DNA replication ,nutritional and metabolic diseases ,General Chemistry ,Cell biology ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Gene Expression Regulation ,Homologous recombination - Abstract
Regulation of end-processing is critical for accurate repair and to switch between homologous recombination (HR) and non-homologous end joining (NHEJ). End resection is a two-stage process but very little is known about regulation of the long-range resection, especially in humans. WRN participates in one of the two alternative long-range resection pathways mediated by DNA2 or EXO1. Here we demonstrate that phosphorylation of WRN by CDK1 is essential to perform DNA2-dependent end resection at replication-related DSBs, promoting HR, replication recovery and chromosome stability. Mechanistically, S1133 phosphorylation of WRN is dispensable for relocalization in foci but is involved in the interaction with the MRE11 complex. Loss of WRN phosphorylation negatively affects MRE11 foci formation and acts in a dominant negative manner to prevent long-range resection altogether, thereby licensing NHEJ at collapsed forks. Collectively, we unveil a CDK1-dependent regulation of the WRN-DNA2-mediated resection and identify an undescribed function of WRN as a DSB repair pathway switch., End-resection of double strand DNA breaks is essential for pathway choice between non-homologous end-joining and homologous recombination. Here the authors show that phosphorylation of WRN helicase by CDK1 is essential for resection at replication-related breaks.
- Published
- 2016
30. Fanconi Anemia: a DNA Repair Disorder Characterized by Accelerated Decline of the Hematopoietic Stem Cell Compartment and Other Features of Aging
- Author
-
Michael M. Seidman, Robert M. Brosh, Marina A. Bellani, and Yie Liu
- Subjects
0301 basic medicine ,Aging ,DNA Repair ,DNA repair ,DNA damage ,Biology ,Biochemistry ,Article ,Epigenesis, Genetic ,03 medical and health sciences ,Fanconi anemia ,medicine ,Humans ,Epigenetics ,Molecular Biology ,Bone marrow failure ,Genetic disorder ,Hematopoietic stem cell ,Telomere ,medicine.disease ,Hematopoietic Stem Cells ,Fanconi Anemia Complementation Group Proteins ,030104 developmental biology ,medicine.anatomical_structure ,Fanconi Anemia ,Neurology ,Immunology ,Biotechnology ,DNA Damage - Abstract
Fanconi Anemia (FA) is a rare autosomal genetic disorder characterized by progressive bone marrow failure (BMF), endocrine dysfunction, cancer, and other clinical features commonly associated with normal aging. The anemia stems directly from an accelerated decline of the hematopoietic stem cell compartment. Although FA is a complex heterogeneous disease linked to mutations in 19 currently identified genes, there has been much progress in understanding the molecular pathology involved. FA is broadly considered a DNA repair disorder and the FA gene products, together with other DNA repair factors, have been implicated in interstrand cross-link (ICL) repair. However, in addition to the defective DNA damage response, altered epigenetic regulation, and telomere defects, FA is also marked by elevated levels of inflammatory mediators in circulation, a hallmark of faster decline in not only other hereditary aging disorders but also normal aging. In this review, we offer a perspective of FA as a monogenic accelerated aging disorder, citing the latest evidence for its multi-factorial deficiencies underlying its unique clinical and cellular features.
- Published
- 2016
31. Biochemical and Cell Biological Assays to Identify and Characterize DNA Helicase Inhibitors
- Author
-
Taraswi Banerjee, Monika Aggarwal, Joshua A. Sommers, and Robert M. Brosh
- Subjects
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
32. DNA Repair and Replication Fork Helicases Are Differentially Affected by Alkyl Phosphotriester Lesion
- Author
-
Daniel L. Kaplan, Zvi Kelman, Yuliang Wu, Stephen Yu, Ting Xu, Avvaru N. Suhasini, Robert M. Brosh, and Joshua A. Sommers
- Subjects
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
33. 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
-
Robert M. Brosh, Monika Aggarwal, Robert H. Shoemaker, and Joshua A. Sommers
- Subjects
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
34. Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes
- Author
-
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
35. Delineation of WRN helicase function with EXO1 in the replicational stress response
- Author
-
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
36. Hitting the bull's eye: Novel directed cancer therapy through helicase-targeted synthetic lethality
- Author
-
Robert M. Brosh and Monika Aggarwal
- Subjects
DNA Repair ,DNA repair ,DNA damage ,medicine.medical_treatment ,Antineoplastic Agents ,Eukaryotic DNA replication ,Synthetic lethality ,Computational biology ,Biochemistry ,Article ,chemistry.chemical_compound ,Drug Delivery Systems ,medicine ,Humans ,Molecular Biology ,Genetics ,biology ,DNA Helicases ,Helicase ,Cancer ,Cell Biology ,medicine.disease ,Radiation therapy ,chemistry ,Drug Design ,biology.protein ,DNA - Abstract
Designing strategies for anti-cancer therapy have posed a significant challenge. One approach has been to inhibit specific DNA repair proteins and their respective pathways to enhance chemotherapy and radiation therapy used to treat cancer patients. Synthetic lethality represents an approach that exploits pre-existing DNA repair deficiencies in certain tumors to develop inhibitors of DNA repair pathways that compensate for the tumor-associated repair deficiency. Since helicases play critical roles in the DNA damage response and DNA repair, particularly in actively dividing and replicating cells, it is proposed that the identification and characterization of synthetic lethal relationships of DNA helicases will be of value in developing improved anti-cancer treatment strategies. In this review, we discuss this hypothesis and current evidence for synthetic lethal interactions of eukaryotic DNA helicases in model systems.
- Published
- 2009
37. Unique and important consequences of RECQ1 deficiency in mammalian cells
- Author
-
Robert M. Brosh and Sudha Sharma
- Subjects
Models, Molecular ,Genetics ,Genome instability ,Premature aging ,DNA Repair ,RecQ Helicases ,Protein Conformation ,DNA damage ,DNA repair ,Helicase ,DNA ,Cell Biology ,Biology ,Article ,Genomic Instability ,Substrate Specificity ,Gene Expression Regulation ,Chromosome instability ,biology.protein ,Humans ,DNA mismatch repair ,Homologous recombination ,Molecular Biology ,Developmental Biology - Abstract
Five members of the RecQ subfamily of DEx-H-containing DNA helicases have been identified in both human and mouse, and mutations in BLM, WRN, and RECQ4 are associated with human diseases of premature aging, cancer, and chromosomal instability. Although a genetic disease has not been linked to RECQ1 mutations, RECQ1 helicase is the most highly expressed of the human RecQ helicases, suggesting an important role in cellular DNA metabolism. Recent advances have elucidated a unique role of RECQ1 to suppress genomic instability. Embryonic fibroblasts from RECQ1-deficient mice displayed aneuploidy, chromosomal instability, and increased load of DNA damage.(1) Acute depletion of human RECQ1 renders cells sensitive to DNA damage and results in spontaneous gamma-H2AX foci and elevated sister chromatid exchanges, indicating aberrant repair of DNA breaks.(2) Consistent with a role in DNA repair, RECQ1 relocalizes to irradiation-induced nuclear foci and associates with chromatin.(2) RECQ1 catalytic activities(3) and interactions with DNA repair proteins(2,4,5) are likely to be important for its molecular functions in genome homeostasis. Collectively, these studies provide the first evidence for an important role of RECQ1 to confer chromosomal stability that is unique from that of other RecQ helicases and suggest its potential involvement in tumorigenesis.
- Published
- 2008
38. Human premature aging, DNA repair and RecQ helicases
- Author
-
Robert M. Brosh and Vilhelm A. Bohr
- Subjects
Genome instability ,Premature aging ,DNA Replication ,Werner Syndrome Helicase ,DNA Repair ,DNA repair ,RecQ helicase ,Cellular homeostasis ,Chromosomal Instability ,Neoplasms ,Replication Protein A ,Genetics ,Humans ,Survey and Summary ,education ,Replication protein A ,education.field_of_study ,biology ,RecQ Helicases ,Helicase ,Aging, Premature ,Telomere ,Exodeoxyribonucleases ,Mutation ,biology.protein ,Sister Chromatid Exchange - Abstract
Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important roles in the DNA damage response. The RecQ family of DNA helicases is of particular interest since several human RecQ helicases are defective in diseases associated with premature aging and cancer. In this review, we will provide an update on our understanding of the specific roles of human RecQ helicases in the maintenance of genomic stability through their catalytic activities and protein interactions in various pathways of cellular nucleic acid metabolism with an emphasis on DNA replication and repair. We will also discuss the clinical features of the premature aging disorders associated with RecQ helicase deficiencies and how they relate to the molecular defects.
- Published
- 2007
39. DNA Repair Helicases as Targets for Anti-Cancer Therapy
- Author
-
Robert M. Brosh and Rigu Gupta
- Subjects
DNA Repair ,DNA repair ,DNA damage ,Antineoplastic Agents ,Biochemistry ,chemistry.chemical_compound ,Neoplasms ,Drug Discovery ,Animals ,Humans ,DNA Breaks, Double-Stranded ,Enzyme Inhibitors ,Pharmacology ,Genetics ,biology ,Organic Chemistry ,DNA Helicases ,Helicase ,Base excision repair ,Combined Modality Therapy ,Double Strand Break Repair ,Cross-Linking Reagents ,DNA Repair Enzymes ,chemistry ,Chemotherapy, Adjuvant ,Drug Design ,biology.protein ,Cancer research ,Molecular Medicine ,RNA Interference ,DNA mismatch repair ,DNA ,Nucleotide excision repair - Abstract
The genetic complexity of cancer has posed a formidable challenge to devising successful therapeutic treatments. Tumor resistance to cytotoxic chemotherapy drugs and radiation which induce DNA damage has limited their effectiveness. Targeting the DNA damage response is a strategy for combating cancer. The prospect for success of chemotherapy treatment may be improved by the selective inactivation of a DNA repair pathway. A key class of proteins involved in various DNA repair pathways is comprised of energy-driven nucleic acid unwinding enzymes known as helicases. DNA helicases have been either implicated or have proposed roles in nucleotide excision repair, mismatch repair, base excision repair, double strand break repair, and most recently cross-link repair. In addition to DNA repair, helicases have been implicated in the cellular processes of replication, recombination, transcription, and RNA stability/processing. The emerging evidence indicates that helicases have vital roles in pathways necessary for the maintenance of genomic stability. In support of this, a growing number of human genetic disorders are attributed to mutations in helicase genes. Because of their essential roles in nucleic acid metabolism, and more specifically the DNA damage response, helicases may be a suitable target of chemotherapy. In this review, we have explored this hypothesis and provided a conceptual framework for combinatorial treatments that might be used for combating cancer by inhibiting helicase function in tumor cells that already have compromised DNA repair and/or DNA damage signaling. This review is focused on helicase pathways, with a special emphasis on DNA cross-link repair and double strand break repair, that impact cancer biology and how cancer cells may be chemosensitized through the impairment of helicase function.
- Published
- 2007
40. Close encounters for the first time: Helicase interactions with DNA damage
- Author
-
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
41. Inhibition of BACH1 (FANCJ) helicase by backbone discontinuity is overcome by increased motor ATPase or length of loading strand
- Author
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Sharon B. Cantor, Kevin M. Doherty, Robert M. Brosh, Joshua A. Sommers, Rigu Gupta, and Sudha Sharma
- Subjects
DNA repair ,ATPase ,Molecular Sequence Data ,DNA, Single-Stranded ,Catalysis ,chemistry.chemical_compound ,Protein structure ,Genetics ,Humans ,BRIP1 Gene ,Amino Acid Sequence ,Adenosine Triphosphatases ,chemistry.chemical_classification ,Polymorphism, Genetic ,Sugar phosphates ,biology ,Nucleic Acid Enzymes ,DNA Helicases ,Helicase ,DNA ,RNA Helicase A ,Fanconi Anemia Complementation Group Proteins ,Protein Structure, Tertiary ,Cell biology ,Basic-Leucine Zipper Transcription Factors ,chemistry ,Biochemistry ,biology.protein - Abstract
The BRCA1 associated C-terminal helicase (BACH1) associated with breast cancer has been implicated in double strand break (DSB) repair. More recently, BACH1 (FANCJ) has been genetically linked to the chromosomal instability disorder Fanconi Anemia (FA). Understanding the roles of BACH1 in cellular DNA metabolism and how BACH1 dysfunction leads to tumorigenesis requires a comprehensive investigation of its catalytic mechanism and molecular functions in DNA repair. In this study, we have determined that BACH1 helicase contacts with both the translocating and the non-translocating strands of the duplex are critical for its ability to track along the sugar phosphate backbone and unwind dsDNA. An increased motor ATPase of a BACH1 helicase domain variant (M299I) enabled the helicase to unwind the backbone-modified DNA substrate in a more proficient manner. Alternatively, increasing the length of the 5′ tail of the DNA substrate allowed BACH1 to overcome the backbone discontinuity, suggesting that BACH1 loading mechanism is critical for its ability to unwind damaged DNA molecules.
- Published
- 2006
42. Cockayne syndrome group B protein has novel strand annealing and exchange activities
- Author
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Martin M. Soerensen, Tina Thorslund, Sudha Sharma, Vilhelm A. Bohr, Meltem Muftuoglu, Tinna Stevnsner, and Robert M. Brosh
- Subjects
Recombination, Genetic ,musculoskeletal diseases ,DNA Repair ,DNA repair ,DNA damage ,DNA Helicases ,DNA, Single-Stranded ,Base excision repair ,Biology ,medicine.disease ,Molecular biology ,Catalysis ,Article ,Cockayne syndrome ,chemistry.chemical_compound ,Adenosine Triphosphate ,chemistry ,Replication Protein A ,Genetics ,medicine ,Phosphorylation ,Homologous recombination ,Replication protein A ,DNA ,Nucleotide excision repair - Abstract
Cockayne syndrome (CS) is a rare inherited human genetic disorder characterized by UV sensitivity, severe neurological abnormalities and prageroid symptoms. The CS complementation group B (CSB) protein is involved in UV-induced transcription coupled repair (TCR), base excision repair and general transcription. CSB also has a DNA-dependent ATPase activity that may play a role in remodeling chromatin in vivo. This study reports the novel finding that CSB catalyzes the annealing of complementary single-stranded DNA (ssDNA) molecules with high efficiency, and has strand exchange activity. The rate of CSB-catalyzed annealing of complementary ssDNA is 25-fold faster than the rate of spontaneous ssDNA annealing under identical in vitro conditions and the reaction occurs with a high specificity in the presence of excess non-homologous ssDNA. The specificity and intrinsic nature of the reaction is also confirmed by the observation that it is stimulated by dephosphorylation of CSB, which occurs after UV-induced DNA damage, and is inhibited in the presence of ATPgammaS. Potential roles of CSB in cooperation with strand annealing and exchange activities for TCR and homologous recombination are discussed.
- Published
- 2006
43. Modulation of Werner Syndrome Protein Function by a Single Mutation in the Conserved RecQ Domain
- Author
-
Jin-Shan Hu, Guang-Xin Lin, Robert M. Brosh, Jae Wan Lee, Kevin M. Doherty, Wen-Hsing Cheng, Cayetano von Kobbe, Wangyong Zeng, Rika Kusumoto, and Vilhelm A. Bohr
- Subjects
Premature aging ,congenital, hereditary, and neonatal diseases and abnormalities ,Werner Syndrome Helicase ,DNA repair ,Amino Acid Motifs ,Molecular Sequence Data ,Mutation, Missense ,In Vitro Techniques ,Biology ,medicine.disease_cause ,Biochemistry ,Cell Line ,medicine ,Holliday junction ,Humans ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Conserved Sequence ,Werner syndrome ,Adenosine Triphosphatases ,Genetics ,Mutation ,Base Sequence ,RecQ Helicases ,Sequence Homology, Amino Acid ,Mutagenesis ,DNA Helicases ,DNA replication ,nutritional and metabolic diseases ,Helicase ,DNA ,Cell Biology ,medicine.disease ,Recombinant Proteins ,Protein Structure, Tertiary ,Exodeoxyribonucleases ,Mutagenesis, Site-Directed ,biology.protein ,Werner Syndrome - Abstract
Naturally occurring mutations in the human RECQ3 gene result in truncated Werner protein (WRN) and manifest as a rare premature aging disorder, Werner syndrome. Cellular and biochemical studies suggest a multifaceted role of WRN in DNA replication, DNA repair, recombination, and telomere maintenance. The RecQ C-terminal (RQC) domain of WRN was determined previously to be the major site of interaction for DNA and proteins. By using site-directed mutagenesis in the WRN RQC domain, we determined which amino acids might be playing a critical role in WRN function. A site-directed mutation at Lys-1016 significantly decreased WRN binding to fork or bubble DNA substrates. Moreover, the Lys-1016 mutation markedly reduced WRN helicase activity on fork, D-loop, and Holliday junction substrates in addition to reducing significantly the ability of WRN to stimulate FEN-1 incision activities. Thus, DNA binding mediated by the RQC domain is crucial for WRN helicase and its coordinated functions. Our nuclear magnetic resonance data on the three-dimensional structure of the wild-type RQC and Lys-1016 mutant proteins display a remarkable similarity in their structures.
- Published
- 2005
44. RECQ1 Helicase Interacts with Human Mismatch Repair Factors That Regulate Genetic Recombination*[boxs]
- Author
-
Teresa M. Wilson, Robert M. Brosh, Laura A. Uzdilla, Kevin M. Doherty, Alessandro Vindigni, Sudha Sharma, and Sheng Cui
- Subjects
Premature aging ,DNA Repair ,DNA repair ,Blotting, Western ,Enzyme-Linked Immunosorbent Assay ,medicine.disease_cause ,Biochemistry ,Genetic recombination ,Proto-Oncogene Proteins ,medicine ,Humans ,Immunoprecipitation ,Molecular Biology ,Gene ,Adenosine Triphosphatases ,Recombination, Genetic ,Genetics ,Mutation ,Genome ,Dose-Response Relationship, Drug ,RecQ Helicases ,biology ,DNA Helicases ,Helicase ,DNA ,Cell Biology ,Recombinant Proteins ,Protein Structure, Tertiary ,DNA-Binding Proteins ,DNA Repair Enzymes ,Exodeoxyribonucleases ,MutS Homolog 2 Protein ,MSH2 ,biology.protein ,DNA mismatch repair ,HeLa Cells ,Protein Binding - Abstract
Understanding the molecular and cellular functions of RecQ helicases has attracted considerable interest since several human diseases characterized by premature aging and/or cancer have been genetically linked to mutations in genes of the RecQ family. Although a human disease has not yet been genetically linked to a mutation in RECQ1, the prominent roles of RecQ helicases in the maintenance of genome stability suggest that RECQ1 helicase is likely to be important in vivo. To acquire a better understanding of RECQ1 cellular and molecular functions, we have investigated its protein interactions. Using a co-immunoprecipitation approach, we have identified several DNA repair factors that are associated with RECQ1 in vivo. Direct physical interaction of these repair factors with RECQ1 was confirmed with purified recombinant proteins. Importantly, RECQ1 stimulates the incision activity of human exonuclease 1 and the mismatch repair recognition complex MSH2/6 stimulates RECQ1 helicase activity. These protein interactions suggest a role of RECQ1 in a pathway involving mismatch repair factors. Regulation of genetic recombination, a proposed role for RecQ helicases, is supported by the identified RECQ1 protein interactions and is discussed.
- Published
- 2005
45. DNA Helicases as Targets for Anti-Cancer Drugs
- Author
-
Kevin M. Doherty, Sudha Sharma, and Robert M. Brosh
- Subjects
DNA Replication ,Cancer Research ,Telomerase ,DNA Repair ,DNA repair ,Antineoplastic Agents ,G-quadruplex ,Substrate Specificity ,chemistry.chemical_compound ,Animals ,Humans ,Enzyme Inhibitors ,Cell Proliferation ,Pharmacology ,Gene knockdown ,biology ,DNA Helicases ,Helicase ,DNA ,Helicase Gene ,Cell biology ,Telomere ,G-Quadruplexes ,chemistry ,biology.protein ,Nucleic Acid Conformation ,Molecular Medicine - Abstract
DNA helicases have essential roles in nucleic acid metabolism by facilitating cellular processes including replication, recombination, DNA repair, and transcription. The vital roles of helicases in these pathways are reflected by their emerging importance in the maintenance of genomic stability. Recently, a number of human diseases with cancer predisposition have been shown to be genetically linked to a specific helicase defect. This has led researchers to further investigate the roles of helicases in cancer biology, and to study the efficacy of targeting human DNA helicases for anti-cancer drug treatment. Helicase-specific inhibition in malignant cells may compromise the high proliferation rates of cancerous tissues. The role of RecQ helicases in response to replicational stress suggests a molecular target for selectively eliminating malignant tumor cells by a cancer chemotherapeutic agent. Alternate DNA secondary structures such as G-quadruplexes that may form in regulatory regions of oncogenes or G-rich telomere sequences are potential targets for cancer therapy since these sequence-specific structures are proposed to affect gene expression and telomerase activation, respectively. Small molecule inhibitors of G-quadruplex helicases may be used to regulate cell cycle progression by modulating promotor activation or disrupting telomere maintenance, important processes of cellular transformation. The design of small molecules which deter helicase function at telomeres may provide a molecular target since telomerase activity is necessary for the proliferation of numerous immortal cells. Although evidence suggests that helicases are specifically inhibited by certain DNA binding compounds, another area of promise in anti-cancer therapy is siRNA technology. Specific knockdown of helicase expression can be utilized as a means to sensitize oncogenic proliferating cell lines. This review will address these topics in detail and summarize the current avenues of research in anti-cancer therapy targeting helicases through small molecule inhibitors of DNA-protein complexes, DNA binding drugs, or down-regulation of helicase gene expression.
- Published
- 2005
46. p53 Modulates RPA-Dependent and RPA-Independent WRN Helicase Activity
- Author
-
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
47. Stimulation of Flap Endonuclease-1 by the Bloom's Syndrome Protein
- Author
-
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
48. Fine-tuning DNA repair by protein acetylation
- Author
-
Sanjay Kumar Bharti and Robert M. Brosh
- Subjects
0301 basic medicine ,Mutation ,Fine-tuning ,DNA repair ,DNA damage ,0206 medical engineering ,02 engineering and technology ,Cell Biology ,Base excision repair ,Biology ,medicine.disease_cause ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Acetylation ,medicine ,DNA mismatch repair ,Molecular Biology ,020602 bioinformatics ,Developmental Biology ,Nucleotide excision repair - Abstract
Post-translational modifications of target DNA repair proteins may help to regulate the mechanism of DNA damage correction. A good analogy is an automobile tune-up necessary to make a car operate a...
- Published
- 2016
49. The transcriptional response after oxidative stress is defective in Cockayne syndrome group B cells
- Author
-
Alfred May, Kevin G. Becker, Vilhelm A. Bohr, Kasper J Kyng, Wen-Hsing Cheng, Robert M. Brosh, and Catheryne Chen
- Subjects
DNA Replication ,Cancer Research ,DNA Repair ,Transcription, Genetic ,DNA damage ,DNA repair ,Recombinant Fusion Proteins ,Mutant ,Biology ,Transfection ,Cockayne syndrome ,Cell Line ,Transcription (biology) ,Genetics ,medicine ,Humans ,Cockayne Syndrome ,Poly-ADP-Ribose Binding Proteins ,Molecular Biology ,Gene ,Cell Line, Transformed ,Oligonucleotide Array Sequence Analysis ,Adenosine Triphosphatases ,Gene Expression Profiling ,Genetic Complementation Test ,DNA Helicases ,Reproducibility of Results ,Hydrogen Peroxide ,Fibroblasts ,Blotting, Northern ,medicine.disease ,Chromatin ,Cell biology ,Complementation ,Oxidative Stress ,DNA Repair Enzymes ,Gene Expression Regulation ,Signal Transduction - Abstract
Cockayne syndrome (CS) is a human hereditary disease belonging to the group of segmental progerias, and the clinical phenotype is characterized by postnatal growth failure, neurological dysfunction, cachetic dwarfism, photosensitivity, sensorineural hearing loss, and retinal degradation. CS-B cells are defective in transcription-coupled DNA repair, base excision repair, transcription, and chromatin structural organization. Using array analysis, we have examined the expression profile in CS complementation group B (CS-B) fibroblasts after exposure to oxidative stress (H2O2) before and after complete complementation with the CSB gene. The following isogenic cell lines were compared: CS-B cells (CS-B null), CS-B cells complemented with wild-type CSB (CS-B wt), and a stably transformed cell line with a point mutation in the ATPase domain of CSB (CS-B ATPase mutant). In the wt rescued cells, we detected significant induction (two-fold) of 112 genes out of the 6912 analysed. The patterns suggested an induction or upregulation of genes involved in several DNA metabolic processes including DNA repair, transcription, and signal transduction. In both CS-B mutant cell lines, we found a general deficiency in transcription after oxidative stress, suggesting that the CSB protein influenced the regulation of transcription of certain genes. Of the 6912 genes, 122 were differentially regulated by more than two-fold. Evidently, the ATPase function of CSB is biologically important as the deficiencies seen in the ATPase mutant cells are very similar to those observed in the CS-B-null cells. Some major defects are in the transcription of genes involved in DNA repair, signal transduction, and ribosomal functions.
- Published
- 2003
50. Functional consequences of mutations in the conserved SF2 motifs and post-translational phosphorylation of the CSB protein
- Author
-
Pia M. Martensen, Vilhelm A. Bohr, Robert M. Brosh, Mette Christiansen, Tinna Stevnsner, and Charlotte Modin
- Subjects
musculoskeletal diseases ,Premature aging ,congenital, hereditary, and neonatal diseases and abnormalities ,DNA Repair ,DNA repair ,DNA damage ,ATPase ,Amino Acid Motifs ,Mutant ,Cockayne syndrome ,Genetics ,medicine ,Humans ,Phosphorylation ,Poly-ADP-Ribose Binding Proteins ,Cells, Cultured ,Conserved Sequence ,Adenosine Triphosphatases ,Base Sequence ,biology ,DNA Helicases ,nutritional and metabolic diseases ,Articles ,DNA ,Base excision repair ,medicine.disease ,Molecular biology ,Chromatin ,DNA Repair Enzymes ,Mutation ,biology.protein ,Nucleic Acid Conformation ,Protein Processing, Post-Translational ,DNA Damage - Abstract
The rare inherited human genetic disorder Cockayne syndrome (CS) is characterized by developmental abnormalities, UV sensitivity and premature aging. The cellular and molecular phenotypes of CS include increased sensitivity to UV-induced and oxidative DNA lesions. Two genes are involved: CSA and CSB. The CS group B (CSB) protein has roles in transcription, transcription-coupled repair, and base excision repair. It is a DNA stimulated ATPase and remodels chromatin in vitro. Here, we have analyzed wild-type (wt) and motif II, V and VI mutant CSB proteins. We find that the mutant proteins display different degrees of ATPase activity deficiency, and in contrast to the in vivo complementation studies, the motif II mutant is more defective than motif V and VI CSB mutants. Furthermore, CSB wt ATPase activity was studied with different biologically important DNA cofactors: DNA with different secondary structures and damaged DNA. The results indicate that the state of DNA secondary structure affects the level of CSB ATPase activity. We find that the CSB protein is phosphorylated in untreated cells and that UV irradiation leads to its dephosphorylation. Importantly, dephosphorylation of the protein in vitro results in increased ATPase activity of the protein, suggesting that the activity of the CSB protein is subject to phosphorylation control in vivo. These observations may have significant implications for the function of CSB in vivo.
- Published
- 2003
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