The rates at which lesions are removed by DNA repair can vary widely throughout the genome, with important implications for genomic stability. To study this, we measured the distribution of nucleotide excision repair (NER) rates for UV-induced lesions throughout the budding yeast genome. By plotting these repair rates in relation to genes and their associated flanking sequences, we reveal that, in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organized within the genome. Furthermore, by comparing genome-wide DNA repair rates in wild-type cells and cells defective in the global genome-NER (GG-NER) subpathway, we establish how this alters the distribution of NER rates throughout the genome. We also examined the genomic locations of GG-NER factor binding to chromatin before and after UV irradiation, revealing that GG-NER is organized and initiated from specific genomic locations. At these sites, chromatin occupancy of the histone ...
To investigate how the nucleotide excision repair initiator XPC locates DNA damage in mammalian cell nuclei we analyzed the dynamics of GFP-tagged XPC. Photobleaching experiments showed that XPC constantly associates with and dissociates from chromatin in the absence of DNA damage. DNA-damaging agents retard the mobility of XPC, and UV damage has the most pronounced effect on the mobility of XPC-GFP. XPC exhibited a surprising distinct dynamic behavior and subnuclear distribution compared with other NER factors. Moreover, we uncovered a novel regulatory mechanism for XPC. Under unchallenged conditions, XPC is continuously exported from and imported into the nucleus, which is impeded when NER lesions are present. XPC is omnipresent in the nucleus, allowing a quick response to genotoxic stress. To avoid excessive DNA probing by the low specificity of the protein, the steady-state level in the nucleus is controlled by nucleus-cytoplasm shuttling, allowing temporally higher concentrations of XPC in ...
Marian Blanca Ramírez from the CSIC in Spain has been studying the effects of LRRK2, a protein associated with Parkinsons disease, on cell motility. A Travelling Fellowship from Journal of Cell Science allowed her to spend time in Prof Maddy Parsons lab at Kings College London, learning new cell migration assays and analysing fibroblasts cultured from individuals with Parkinsons. Read more on her story here. Where could your research take you? The deadline to apply for the current round of Travelling Fellowships is 23rd Feburary 2018. Apply now!. ...
It has been suggested that phosphorylation of the histone variant H2AX after ultraviolet light (UV) irradiation is triggered by DNA double-strand breaks induced as replication forks collide with UV-induced bulky lesions. More recently, it has been shown that UV-induced H2AX phosphorylation can also occur outside of S-phase, but the mechanism for this replication-independent induction is not well understood. In this study, we show that H2AX phosphorylation after UV irradiation is triggered by DNA repair intermediates and is induced in all phases of the cell cycle. Accumulation of DNA repair intermediates by inhibition of DNA repair synthesis resulted in a marked increase of H2AX phosphorylation in repair proficient but not repair-deficient xeroderma pigmentosum-A cells. Using chemical inhibitors of the PI(3)-like kinase family of protein kinases as well as ataxia telangiectasia mutated and Rad-3 related (ATR)-deficient Seckel syndrome cells and ataxia telangiectasia mutated-deficient ataxia ...
The expression of chloramphenicol acetyl transferase (CAT) as reporter can be detected in a variety of ways and has been the reporter of choice in reporter-vector systems for long time [20]. However, the notable disadvantage of these systems, however, is that the expressed reporter protein must be assayed via enzymatic reactions using whole cell extracts into which the reporter vector has been transfected [21].. Furthermore the quantification of DNA repair capacity via protein expression is disadvantageous because cells normally take up more than one plasmid, and therefore it is not possible to quantify the number of cells that are able to repair. With a view to the development of (skin-) malignancies, we also have to address the question whether a decrease in DNA repair capacity of a cell population rests on complete repair deficiency of single cells (while others remained unaffected) or on a partial decrease of DNA repair capacity in all cells. In our view single, repair deficient cells ...
TY - JOUR. T1 - E2F1 regulates the base excision repair gene XRCC1 and promotes DNA repair. AU - Chen, Dexi. AU - Yu, Zhiyong. AU - Zhu, Zhiyi. AU - Lopez, Charles D.. PY - 2008/5/30. Y1 - 2008/5/30. N2 - The E2F1 transcription factor activates S-phase-promoting genes, mediates apoptosis, and stimulates DNA repair through incompletely understood mechanisms. XRCC1 (x-ray repair cross-complementing group 1) protein is important for efficient single strand break/base excision repair. Although both damage and proliferative signals increase XRCC1 levels, the mechanisms regulating XRCC1 transcription remain unclear. To study these upstream mechanisms, the XRCC1 promoter was cloned into a luciferase reporter. Ectopic expression of wild-type E2F1, but not an inactive mutant E2F1(132E), activated the XRCC1 promoter-luciferase reporter, and deletion of predicted E2F1 binding sites in the promoter attenuated E2F1-induced activation. Endogenous XRCC1 expression increased in cells conditionally expressing ...
DNA repair plays a key role in carcinogenesis through the removal and repair of DNA damage induced by endogenous and environmental sources. The DNA repair system included four pathways: 1) Base Excision Repair (BER), 2) Nucleotide Excision Repair (NER), 3) Mismatch Repair (MMR) and 4) Double-Strand Break Repair, including homologous recombination pathway and nonhomologous end-joining repair pathway. Decreased and impaired DNA repair capacity has been reported in various cancers, however, its effect on prostate cancer still under investigated.. Common polymorphisms in DNA repair gene may alter protein function and individuals capacity to repair damaged DNA, hence, influence the cancer susceptibility. Polymorphic variants of DNA repair gene have been found to be associated with cancer susceptibility, but rare studies have investigated their effect on prostate cancer. Since variation in the function of these DNA repair genes also impact a cancer cells viability or resistance to treatment, genetic ...
Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC(-/-) background. XPC(-/-) cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA
The purpose of this study was to elucidate if the cellular DNA repair capacity, genotypes of DNA repair genes and the occurrence of breast cancer can be associated with each other on the same set of probands in a single study. Rarely characterized features of the DNA repair capacity measures were studied as a prerequisite for their use in the definition as an intermediate phenotype of cancer. The study population comprised different cohorts with sporadic or familial breast cancer cases and controls. DNA repair assays were performed in parallel on peripheral blood samples of the probands: the baseline and the radiation induced micronucleus (MN) assay, the baseline and the induced sister chromatid exchange (SCE) assay and the mitotic delay (MD) assay. 23 missense variants in 15 genes were genotyped. An increased induced MN frequency and a decreased MD index were associated with the occurrence of breast cancer. The minor allele of the variant Slx4 S1271F (rs3810813) was enriched in breast cancer ...
Oxidized DNA base lesions, such as thymine glycol (Tg) and 8-hydroxyguanine, are often toxic and mutagenic and have been implicated in carcinogenesis. To clarify whether NEIL1 protein, which exhibits excision repair activity towards such base lesions, is involved in gastric carcinogenesis, we examined 71 primary gastric cancers from Japanese patients and four gastric cancer cell lines for mutations and genetic polymorphisms of the NEIL1 gene. We also examined 20 blood samples from Chinese patients for NEIL1 genetic polymorphisms. Three mutations (c.82_84delGAG:p.Glu28del, c.936G , A and c.1000A , G:p.Arg334Gly) and two genetic polymorphisms were identified. When the excision repair activity towards double-stranded oligonucleotide containing a Tg:A base pair was compared among six types of recombinant NEIL1 proteins, p.Glu28del-type NEIL1, found in a primary case, was found to exhibit an extremely low activity level. Moreover, c.936G , A, located in the last nucleotide of exon 10 and detected in ...
Elliott, R, Astley, S, Southon, S and Archer, D (2000) The development of DNA repair assays which show that dietary carrots stimulate DNA repair activity In: 3rd Conference on Food and Cancer Prevention, 1999-09-05 - 1999-09-08, UNIV E ANGLIA, NORWICH, ENGLAND. Full text not available from this repository ...
DNA repair capacity varies greatly between individuals, and evidence has begun to link this variation to cancer risk, obesity and related chronic diseases. There is also emerging evidence that dietary components can affect DNA repair, but research to date has been restricted by methods for measuring DNA repair. This study made use of newly developed microplate-based assays for the direct determination of DNA repair enzyme activities. Lipid loading of the HepG2 human hepatocellular carcinoma cell line was employed as a model to test the hypothesis that hepatic steatosis affects DNA repair activity via induction of oxidative stress.. ...
Purpose: DNA repair deficiencies have been postulated to play a role in the development and progression of cardiovascular disease (CVD). The hypothesis is that DNA damage may induce cell death that promotes formation of unstable plaques. Defects in the DNA repair mechanisms may therefore increase the risk of cardiovascular events. Using a DNA repair pathway approach we systematically examined whether common genetic variation in DNA repair genes influence the risk of cardiovascular disease progression and cardiovascular events.. Methods: DNA repair genes were selected using a systematic pathway approach and a total of 59 single nucleotide polymorphisms (SNPs) within 24 genes were selected using several criteria such as adequate heterogeneity, LD pattern and validation status. Eleven additional SNPs from literature were added. Genotyping was performed using Illumina Human 660-Quad Beadchips in 5244 subject of the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER) study. Collected ...
TY - JOUR. T1 - Polymorphisms in DNA repair genes, smoking, and pancreatic adenocarcinoma risk. AU - Mc Williams, Robert R. AU - Bamlet, William R.. AU - Cunningham, Julie M. AU - Goode, Ellen L. AU - De Andrade, Mariza. AU - Boardman, Lisa Allyn. AU - Petersen, Gloria M. PY - 2008/6/15. Y1 - 2008/6/15. N2 - Base excision repair and nucleotide excision repair are vital responses to multiple types of DNA damage, including damage from tobacco exposure. Single-nucleotide polymorphisms (SNP)in these pathways may affect DNA repair capacity and therefore influence risk for cancer development. We performed a clinic-based, case-control study comprising 481 consecutive patients with confirmed pancreatic adenocarcinoma and 625 healthy controls. Allele and genotype frequencies for 16 SNPs in DNA repair genes ERCC1, XPD/ERCC2, XPC, XPF/ERCC4, OGG1, and XRCC1 were compared after adjusting for age, sex, and smoking history. Subgroup analysis by sex and smoking history was performed. Carriers of one or two ...
Nucleotide excision repair (NER) is a major DNA repair pathway in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation or bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to three genetic disorders that result in predisposition to cancers, accelerated aging, neurological and developmental defects. During NER, more than 30 polypeptides cooperate to recognize, incise, and excise a damaged oligonucleotide from the genomic DNA. Recent papers reveal an additional and unexpected role for the NER factors. In the absence of a genotoxic attack, the promoters of RNA polymerases I- and II-dependent genes recruit XPA, XPC, XPG, and XPF to initiate gene expression. A model that includes the growth arrest and DNA damage 45alpha protein (Gadd45alpha) and the NER factors, in order to maintain the promoter of active genes under a hypomethylated state, has been proposed but remains controversial.
The researchers found that single mismatched ribonucleotides in chromosomal DNA were removed by either the mismatch repair system or RNase H type 2. Mismatched ribonucleotides in the middle of at least four other ribonucleotides required RNase H type 1 for removal.. "We were excited to find that a DNA repair mechanism like mismatch repair was activated by RNA/DNA mismatches and could remove ribonucleotides embedded in chromosomal DNA," explained Storici. "In future studies, we plan to test whether other DNA repair mechanisms, such as nucleotide-excision repair and base-excision repair, can also locate and remove ribonucleotides in DNA.". Using gene correction assays driven by short nucleic acid polymers called oligonucleotides, the researchers showed that when ribonucleotides embedded in DNA were not removed, they served as templates for DNA synthesis and produced a mutation in the DNA. If both the mismatch repair system and RNase H repair mechanisms are disabled, ribonucleotide-driven gene ...
Base excision repair (BER) is the predominant DNA damage repair pathway for the processing of small base lesions, derived from oxidation and alkylation damages. BER is normally defined as DNA repair initiated by lesion-specific DNA glycosylases and completed by either of the two sub-pathways: short-patch BER where only one nucleotide is replaced and long-patch BER where 2-13 nucleotides are replaced. Each sub-pathway of BER relies on the formation of protein complexes that assemble at the site of the DNA lesion and facilitate repair in a coordinated fashion. This process of complex formation appears to provide an increase in specificity and efficiency to the BER pathway, thereby facilitating the maintenance of genome integrity by preventing the accumulation of highly toxic repair intermediates ...
Base excision repair (BER) is the predominant DNA damage repair pathway for the processing of small base lesions, derived from oxidation and alkylation damages. BER is normally defined as DNA repair initiated by lesion-specific DNA glycosylases and completed by either of the two sub-pathways: short-patch BER where only one nucleotide is replaced and long-patch BER where 2-13 nucleotides are replaced. Each sub-pathway of BER relies on the formation of protein complexes that assemble at the site of the DNA lesion and facilitate repair in a coordinated fashion. This process of complex formation appears to provide an increase in specificity and efficiency to the BER pathway, thereby facilitating the maintenance of genome integrity by preventing the accumulation of highly toxic repair intermediates ...
The molecular etiology of breast cancer has proven to be re- markably complex. Most individual oncogenes are disregulated in only approximately 30% of breast tumors, indicating that either very few molecular alterations are common to the majority of breast cancers, or that they have not yet been identified. In striking contrast, we now show that 19 of 19 stage I breast tumors tested with the functional unscheduled DNA synthesis assay exhibited a significant deficiency of DNA nucleotide excision repair (NER) capacity relative to normal epithelial tissue from disease-free controls (n = 23). Loss of DNA repair capacity, including the complex, damage-comprehensive NER pathway, results in genomic instabil- ity, a hallmark of carcinogenesis. By microarray analysis, mRNA ex- pression levels for 20 canonical NER genes were reduced in repre- sentative tumor samples versus normal. Significant reductions were observed in 19 of these genes analyzed by the more sensitive method of RNase protection. These results
The nucleotide excision repair (NER) pathway operates through two sub-pathways: global genome repair (ggNER) and transcription-coupled repair (TCR) or gene- and strand-specific DNA repair [1, 2, 4]. The ggNER is a repair mechanism which has the ability to repair DNA damage to the overall genome with equivalent efficiency. In contrast, TCR is a kind of heterogeneous DNA repair, where repair to the damaged DNA in the status of transcription activity is superior to the silenced genes and the repair of the transcribed strand is superior to the untranscribed strand. Some DNA repair proteins and transcription factors have been identified to be involved in TCR such as CSA, CSB, XPG, XAB2, RNA polymerase II, and TFIIH [1, 7, 8, 24]. Blockage of RNA polymerase □ at the DNA damage site is believed to create a conducive environment for DNA repair [7, 9]. In this report, we provide evidence to demonstrate that DNA-PKcs, a known critical component in the NHEJ pathway of DNA double-strand breaks, is also ...
The role of nucleotide excision repair (NER) in the maintenance of DNA integrity under oxidative assault has yet to be elucidated. A defective NER can result in Xeroderma Pigmentosa (XP) or Cockayne Syndrome (CS), both autosomal recessive diseases, presenting with increased cancer risk and segmental progeria. Although the NER is characterized to be involved in repairing UV-induced damage, it is difficult to attribute all the symptoms of XP and CS to UV-damage. Oxidative stress is thus likely to be an important factor. Other DNA repair proteins including a component of the NER pathway, XPF, have been reported to be involved in telomere dynamics. As the importance of the NER pathway in removing oxidative stress-induced DNA lesions is still unclear, we sought to understand the role of NER in oxidative stress-induced damage protection and telomere-mediated chromosome integrity. In our study, we utilized primary cells derived from patients suffering from XP (XP-A and XP-D) and CS Type II (CS-B), as ...
Efficient and correct repair of DNA damage, especially DNA double-strand breaks (DSBs), is vital for the survival of individual cells and organisms. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. The repair of DSBs in cell lines with different DSB rejoining capabilities was studied after exposure to ionising radiation. A new cell lysis protocol performed at 0ºC, which prevents the inclusion of non-true DSBs in the quantification of DSBs by pulsed-field gel electrophoresis (PFGE), was developed. Results showed that when the standard protocol at 50ºC was used, 30-40% of the initial yield of DSBs corresponds to artifactual DSBs. The lesions transformed to DSBs during incubation at 50ºC were repaired within 60-90 minutes in vivo and the repair was independent of DNA-PK, XRCC1 and PARP-1.. Non-homologous end-joining (NHEJ) is the major DSB repair pathway in mammalian cells. We show that DSBs are processed into long single-stranded DNA (ssDNA) ...
In this study, we report the role of genetic polymorphisms XRCC1 Arg194Trp (C→T) and Arg399Gln (G→A), OGG1 Ser326Cys (C→G), APEX1 Asp148Glu (T→G), MUTYH Gln335His (G→C) and PARP1 Val762Ala (T→C) on the individual susceptibility for TC. The frequencies of the different genotypes observed in the control population are similar to those reported in other Caucasian populations.. XRCC1 is a nuclear protein that, despite lacking any enzymatic activity, plays an important role in the efficient repair of SSBs and in the BER pathway: it acts as a scaffold protein that facilitates the recruitment of multiple DNA repair enzymes (such as Pol β, hOGG1, APEX1, PARP1 and LIG3) to lesion sites and coordinates the DNA damage repair response. Arg194Trp and Arg399Gln are among the most extensively studied XRCC1 coding region SNPs. Both polymorphisms have been shown to alter the functional activity of the resulting protein in vitro and to interfere with cancer susceptibility: the 194Trp allele has been ...
DESCRIPTION (provided by applicant): This is an effort to relate polymorphisms in DNA repair to incidence of breast cancer. Mutations in both the BER and HRR pathways are proposed to make an individual more sensitive to ionizing radiation and breast cancer. Assays for assessing decreased activity in these pathways will also be pursued. Our long-term goal in cancer prevention is to identify high-risk populations by using DNA repair markers and to design mechanistic-based prevention strategy tailored to the specific risk profiles of individuals. Human cells are constantly exposed to exogenous and endogenous carcinogens capable of causing DNA damage. Efficient repair is critical in maintaining genome integrity and genetic variability in DNA repair may influence individuals susceptibility to cancer. Our working hypothesis is that polymorphism of DNA repair genes with nonconservative amino acid substitution may lead to altered protein function in DNA repair and elevated breast cancer risk. Various ...
DNA excision repair protein ERCC-1 is a protein that in humans is encoded by the ERCC1 gene. Together with ERCC4, ERCC1 forms the ERCC1-XPF enzyme complex that participates in DNA repair and DNA recombination. Many aspects of these two gene products are described together here because they are partners during DNA repair. The ERCC1-XPF nuclease is an essential activity in the pathway of DNA nucleotide excision repair (NER). The ERCC1-XPF nuclease also functions in pathways to repair double-strand breaks in DNA, and in the repair of "crosslink" damage that harmfully links the two DNA strands. Cells with disabling mutations in ERCC1 are more sensitive than normal to particular DNA damaging agents, including ultraviolet (UV) radiation and to chemicals that cause crosslinking between DNA strands. Genetically engineered mice with disabling mutations in ERCC1 have defects in DNA repair, accompanied by metabolic stress-induced changes in physiology that result in premature aging. Complete deletion of ...
The drug shows high potency and activity in various experimental models including P-gp- and MRP1-expressing multidrug resistant tumors as well as tumors resistant to platinum derivatives, alkylating agents and topoisomerase I and II inhibitors. Nemorubicin has a peculiar mechanism of action requiring NER activity for its full cytotoxicity. Cell lines defective in NER, which show an increased sensitivity to classical alkylating agents, display a three-four fold resistance to nemorubicin. Interestingly, L1210 murine leukemia cells selected for resistance to nemorubicin (L1210/MMDX) show a collateral sensitivity to both platinum derivatives and alkylating agents and are more sensitive (about 4-5 times) to UV light. In addition, these cells are not able to repair UV-induced damage on transfected DNA (host -cell reactivation assay), suggesting that the NER system might be involved in mediating the cytotoxic activity of nemorubicin. The aim of the present study was to evaluate the possible presence of ...
Explorers have dreamt for centuries of a Fountain of Youth, with healing waters that rejuvenate the old and extend life indefinitely.. Researchers at the University of Rochester, however, have uncovered more evidence that the key to longevity resides instead in a gene.. In a new paper published in the journal Cell, the researchers - including Vera Gorbunova and Andrei Seluanov, professors of biology; Dirk Bohmann, professor of biomedical genetics; and their team of students and postdoctoral researchers - found that the gene sirtuin 6 (SIRT6) is responsible for more efficient DNA repair in species with longer lifespans. The research illuminates new targets for anti-aging interventions and could help prevent age-related diseases.. INEVITABLE DOUBLE-STRAND BREAKS. As humans and other mammals grow older, their DNA is increasingly prone to breaks, which can lead to gene rearrangements and mutations - hallmarks of cancer and aging. For that reason, researchers have long hypothesized that DNA repair ...
Anthracyclines have been widely used as antitumor agents, playing a crucial role in the successful treatment of many types of cancer, despite some side effects related to cardiotoxicity. New anthracyclines have been designed and tested, but the first ones discovered, doxorubicin and daunorubicin, continue to be the drugs of choice. Despite their extensive use in chemotherapy, little is known about the DNA repair mechanisms involved in the removal of lesions caused by anthracyclines. the anthracycline cosmomycin D is the main product isolated from Streptomyces olindensis, characterized by a peculiar pattern of glycosylation with two trisaccharide rings attached to the A ring of the tetrahydrotetracene.We assessed the induction of apoptosis (Sub-G(1)) by cosmomycin D in nucleotide excision repair-deficient fibroblasts (XP-A and XP-C) as well as the levels of DNA damage (alkaline comet assay).Treatment of XP-A and XP-C cells with cosmomycin D resulted in apoptosis in a time-dependent manner, with ...
Eukaryotic cells deploy overlapping repair pathways to resolve DNA damage. Advancements in genome editing take advantage of these pathways to produce permanent genetic changes. Despite recent improvements, genome editing can produce diverse outcomes that can introduce risks in clinical applications. Although homology-directed repair is attractive for its ability to encode precise edits, it is particularly difficult in human cells. Here we discuss the DNA repair pathways that underlie genome editing and strategies to favour various outcomes. Harnessing DNA repair pathways in genome editing In this Review, Yeh, Richardson and Corn discuss the DNA repair pathways that underlie genome editing and recent improvements and strategies to yield desired genomic alterations in cells and organisms.
Mutations caused by DNA damage are a main driver of cancer. We discovered that recognition of newly synthesised histone H4 directs breast cancer type 1 susceptibility protein (BRCA1) to post-replicative chromatin. The switch from mutagenic to error-free DNA double strand break repair by homologous recombination is therefore controlled by chromatin. ...
Following DNA damage, mRNA levels decrease, reflecting a coordinated interaction of the DNA repair, transcription and RNA processing machineries. In this study, we provide evidence that transcription and polyadenylation of mRNA precursors are both affected in vivo by UV treatment. We next show that the polyadenylation factor CstF, plays a direct role in the DNA damage response. Cells with reduced levels of CstF display decreased viability following UV treatment, reduced ability to ubiquitinate RNA polymerase II (RNAP II), and defects in repair of DNA damage. Furthermore, we show that CstF, RNAP II and BARD1 are all found at sites of repaired DNA. Our results indicate that CstF plays an active role in the response to DNA damage, providing a link between transcription-coupled RNA processing and DNA repair.
Inborn defects in DNA repair mechanisms are associated with cancer, aging but also complex metabolic and endocrine disorders. Integrity of the genome is critical for normal cellular function but the DNA is continually challenged by intrinsic and extrinsic genotoxic factors. To counteract DNA damage, cells have evolved DNA repair mechanisms ensuring that the genome remains functionally intact and is faithfully transmitted to progeny. Nucleotide excision repair (NER) is a major DNA repair mechanism that cells employ to remove a wide class of bulky, DNA-distorting lesions from the genome. The importance of NER defects in man is illustrated by rare syndromes that either show increased cancer predisposition or dramatic features of accelerated aging, including depletion of fat depots. However, with the exception of cancer and aging, the links between defects in NER and the rapid onset of developmental defects in humans are not well understood.. Research carried out at the Institute of Molecular ...
Required for radiation resistance and meiotic viability and presumably acts in recombination and recombinational DNA repair pathways.
Mono- and Stereopictres of 5.0 Angstrom coordination sphere of Magnesium atom in PDB 1cw0: Crystal Structure Analysis of Very Short Patch Repair (Vsr) Endonuclease in Complex With A Duplex Dna
Efficient and correct repair of DNA damage, especially DNA double-strand breaks (DSBs), is vital for the survival of individual cells and organisms. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. The repair of DSBs in cell lines with different DSB rejoining capabilities was studied after exposure to ionising radiation. A new cell lysis protocol performed at 0ºC, which prevents the inclusion of non-true DSBs in the quantification of DSBs by pulsed-field gel electrophoresis (PFGE), was developed. Results showed that when the standard protocol at 50ºC was used, 30-40% of the initial yield of DSBs corresponds to artifactual DSBs. The lesions transformed to DSBs during incubation at 50ºC were repaired within 60-90 minutes in vivo and the repair was independent of DNA-PK, XRCC1 and PARP-1.. Non-homologous end-joining (NHEJ) is the major DSB repair pathway in mammalian cells. We show that DSBs are processed into long single-stranded DNA (ssDNA) ...
Kim, M.; Kang, H-Gyoung.; Lee, S.Yup.; Lee, H.Cheol.; Lee, E.Bae.; Choi, Y.Young.; Lee, W.Kee.; Cho, S.; Jin, G.; Jheon, H-Sung.; Son, J.Woong.; Lee, M-Hoon.; Jung, D.Kju.; Cha, S.Ick.; Kim, C.Ho.; Kang, Y.Mo.; Kam, S.; Jung, T.Hoon.; Jheon, S.; Park, J.Yong., 2010: Comprehensive analysis of DNA repair gene polymorphisms and survival in patients with early stage non-small-cell lung cancer
Grain counting by eye is a tedious and time-consuming technique but one with great potential in cell kinetics and for the study of DNA excision repair activity (unscheduled DNA synthesis or UDS). We have been investigating the levels of UDS in human skin sections exposed in situ to ultraviolet radiation using a short-term incubation in tritiated thymidine and autoradiography and the decline in UDS levels with time (repair kinetics). We have adapted an automated image analysis system automatically to assess the number of grains over epidermal cell nuclei in autoradiographs of sections of epidermis. An excellent correlation was observed between visual counting and machine measurement of the area (in pixels) occupied by silver grains. The levels of UDS declined with time as lesions are progressively repaired. The half time (+/- standard deviation) for the reduction in UDS is 7.25 +/- 0.18 h. The grain counts can be significantly increased by increasing the autoradiographic exposure, by increasing ...
Instability: Mutation and DNA repair Mutations DNA repair Instability: Mutation and DNA repair Substitution rates vary throughout the genome. Instability: Mutation and DNA
TY - JOUR. T1 - DNA repair gene variants in relation to overall cancer risk. T2 - A population-based study. AU - Alberg, Anthony J.. AU - Jorgensen, Timothy J.. AU - Ruczinski, Ingo. AU - Wheless, Lee. AU - Shugart, Yin Yao. AU - Berthier-Schaad, Yvette. AU - Kessing, Bailey. AU - Hoffman-Bolton, Judith. AU - Helzlsouer, Kathy J.. AU - Linda Kao, W. H.. AU - Francis, Lesley. AU - Alani, Rhoda M.. AU - Smith, Michael W.. AU - Strickland, Paul Timothy. PY - 2013/1. Y1 - 2013/1. N2 - The hypothesis that germ-line polymorphisms in DNA repair genes influence cancer risk has previously been tested primarily on a cancer site-specific basis. The purpose of this study was to test the hypothesis that DNA repair gene allelic variants contribute to globally elevated cancer risk by measuring associations with risk of all cancers that occurred within a population-based cohort. In the CLUE II cohort study established in 1989 in Washington County, MD, this study was comprised of all 3619 cancer cases ...
Crosslinked DNA is repaired in cells by a combination of enzymes and other factors from the nucleotide excision repair (NER) pathway, homologous recombination, and the base excision repair (BER) pathway. To repair interstrand crosslinks in eukaryotes, a 3 flap endonuclease from the NER, XPF-ERCC1, is recruited to the crosslinked DNA, where it assists in unhooking the DNA by cleaving the 3 strand at the crosslink site. The 5 strand is then cleaved, either by XPF-ERCC1 or another endonuclease, forming a double-strand break (DSB), which can then be repaired by the homologous recombination pathway.[17] DNA crosslinks generally cause loss of overlapping sequence information from the two strands of DNA. Therefore, accurate repair of the damage depends on retrieving the lost information from an undamaged homologous chromosome in the same cell. Retrieval can occur by pairing with a sister chromosome produced during a preceding round of replication. In a diploid cell retrieval may also occur by ...
Gorbunova and her team were able to make their observations by working with genetically-modified mice whose cells produce green fluorescent protein (GFP) that glows each time the breaks are repaired. By tracking how many cells glowed green in different tissues, the researchers determined the efficiency of repair.. "We showed two things with these genetically-modified mice," said Gorbunova. "Not only did the efficiency of DNA repair decline with age, but the mice began using a sloppier repair mechanism, leading to more mutations, particularly in the heart and lungs.". DNA breaks occur frequently because animal cells are under constant assault from routine activities in the environment-whether by a blast of X-rays from a visit to the doctor or simply breathing in oxygen-and, as a result, the DNA molecules often get damaged.. Using the genetically modified mice, the research team can now look at how diet, medicines, and different genetic factors also affect DNA repair in mice.. "These mice may very ...
Defects in cellular DNA repair processes have been linked to genome instability, heritable cancers, and premature aging syndromes. Yet defects in some repair processes manifest themselves primarily in neuronal tissues. This review focuses on studies defining the molecular defects associated with several human neurological disorders, particularly ataxia with oculomotor apraxia 1 (AOA1) and spinocerebellar ataxia with axonal neuropathy 1 (SCAN1). A picture is emerging to suggest that brain cells, due to their nonproliferative nature, may be particularly prone to the progressive accumulation of unrepaired DNA lesions.
17. Katagiri, T., Saito, H., Shinohara, A., Ogawa, H., Kamada, N., Nakamura Y. and Y. Miki. Multiple possible sites of BRCA2 interacting with DNA repair protein Rad51. Genes, Chromosomes and Cancer, 21, 217-222. 1998, CI=43.. 18. Gasior, S., Wang, A., Kohra, Y., Shinohara, A. and D.K. Bishop. Rad52 associates with RPA and functions with Rad55 and Rad57 to assemble meiotic recombination complexes. Genes & Dev., 12, 2208-2221, 1998, CI=183.. 19. Bishop, D.K., Ear, U., Bhattacharyya, A., Calderone, C., Beckett, M., Weichselbaum, R. and A. Shinohara. Xrcc3 is required for assembly of Rad51-complexes in vivo. J. Biol. Chem., 273, 21482-21488. 1998, CI=213.. 20. Takata, M., Sasaki, M., Sonoda, E., Morrison, C., Hashimoto, M., Utsumi, H., Yamaguchi-Iwai, Y., Shinohara, A., and S. Takeda. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in maintenance of chromosome integrity. EMBO J., 17, 5497-5508. 1998, CI=797.. 21. Nishitani, ...
DSB repair pathways. There are three DNA DSB repair pathways: single-strand annealing (SSA), homologous recombination (HR), and nonhomologous end-joining (NHEJ) (51). In SSA, which requires homologous sequences to flank the break site, the nonhomologous DNA ends are removed, leaving overhangs that are aligned and annealed (45). After annealing, DNA synthesis and ligation occur. SSA results in deletion of the region between homologous sequences. In contrast, HR is a relatively error-free mechanism for DSB repair that relies on the homologous region on the sister chromatid as a template for DNA synthesis (Figure 6A) (1;16). HR is limited to replicating cells (including early embryonic developmental stages) and can be observed only after DNA replication in the S and G2 phases (52) . Finally, NHEJ is a rapid repair mechanism that simply ligates broken DNA ends after minimal or no processing (Figure 6B). NHEJ occurs in all phases of the cell cycle and is thought to be the primary DNA repair pathway ...
FUNCTION: Component of the post-replicative DNA mismatch repair system (MMR). Heterodimerizes with MSH2 to form MutS alpha, which binds to DNA mismatches thereby initiating DNA repair. MSH6 provides substrate-binding and substrate-specificity to the complex. When bound, MutS alpha bends the DNA helix and shields approximately 20 base pairs. Acts mainly to repair base-base and single insertion-deletion mismatches that occur during replication, but can also repair longer insertion-deletion loops (IDLs), although with decreasing efficiency as the size of the extrahelical loop increases. After mismatch binding, forms a ternary complex with the MutL alpha heterodimer, which is thought to be responsible for directing the downstream MMR events, including strand discrimination, excision, and resynthesis. ATP binding and hydrolysis by the MutS alpha complex is crucial for MMR. Both subunits bind ATP, but with differing affinities, and their ATPase kinetics are also very different. MSH6 binds and ...
Following the irradiation of non-dividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such "two-strand" mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in non-dividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds, and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32 and Rad5) and in the early steps of the DNA-damage ...
Cisplatin is a crucial agent in the treatment of many solid tumors, yet many tumors have either acquired or intrinsic resistance to the drug. We have used the homozygous diploid deletion pool of Saccharomyces cerevisiae, containing 4728 strains with individual deletion of all nonessential genes, to systematically identify genes that when deleted confer sensitivity to the anticancer agents cisplatin, oxaliplatin, and mitomycin C. We found that deletions of genes involved in nucleotide excision repair, recombinational repair, postreplication repair including translesional synthesis, and DNA interstrand cross-link repair resulted in sensitivity to all three of the agents, although with some differences between the platinum drugs and mitomycin C in the spectrum of required translesional polymerases. Putative defective repair of oxidative damage (imp2Delta strain) also resulted in sensitivity to platinum and oxaliplatin, but not to mitomycin C. Surprisingly in light of their different profiles of ...
Nucleotide excision repair is a DNA repair mechanism. DNA constantly requires repair due to damage that can occur to bases from a vast variety of sources including chemicals but also ultraviolet (UV) light from the sun
BACKGROUND: Several genes encoding for DNA repair molecules implicated in maintaining genomic integrity have been proposed as cancer-susceptibility genes. Although efforts have been made to create synopses for specific fields that summarize the data from genetic association studies, such an overview is not available for genes involved in DNA repair. METHODS: We have created a regularly updated database of studies addressing associations between DNA repair gene variants (excluding highly penetrant mutations) and different types of cancer. Using 1087 datasets and publicly available data from genome-wide association platforms, meta-analyses using dominant and recessive models were performed on 241 associations between individual variants and specific cancer types that had been tested in two or more independent studies. The epidemiological strength of each association was graded with Venice criteria that assess amount of evidence, replication, and protection from bias. All statistical tests were ...
Modern light microscopical techniques were employed to follow dynamical nuclear processes during the cell cycle and during DNA-repair. Laser-UVA-microirradiation The protein Rad51 is essential for the repair of double-strand breaks (DSBs) via the conservative homologous recombination repair pathway. To test the hypothesis that Rad51 localizes to damaged sites during DSB repair, a laser-UVA-microirradiation system was established. With this system spots with sizes around 1 µm in nuclei of living cells can be irradiated with UVA-light. After sensitization of cells by incorporation of BrdU into nuclear DNA and staining with the live cell dye Hoechst 33258, the system can be used to introduce double-strand breaks and single-strand breaks in the irradiated spots. The response of Rad51 to microirradiation By use of laser-UVA microirradiation the localization of Rad51 at damaged sites containing DNA double-strand breaks could be demonstrated. The accumulation of Rad51 at microirradiated sites was ...