XRCC1 and DNA strand break repair

DNA single-strand breaks can arise indirectly, as normal intermediates of DNA base excision repair, or directly from damage to deoxyribose. Because single-strand breaks are induced by endogenous reactive molecules such as reactive oxygen species, these lesions pose a continuous threat to genetic integrity. XRCC1 protein plays a major role in facilitating the repair of single-strand breaks in mammalian cells, via an ability to interact with multiple enzymatic components of repair reactions. Here, the protein-protein interactions facilitated by XRCC1, and the repair processes in which these interactions operate, are reviewed. Models for the repair of single-strand breaks during base excision repair and at direct breaks are presented.     

Genetic polymorphisms of DNA repair gene XRCC1 and risk of uterine leiomyoma

The objective was to study the relationship between the polymorphisms of the DNA repair gene XRCC1 Arg399Gln, Arg194Trp, and Arg280His uterine leiomyoma in a Chinese population. In the case–control study, we compared the XRCC1 gene polymorphism of 136 uterine leiomyoma patients and 140 healthy controls by using the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). The results suggested that the genotype Arg/Arg of codon 280 was significantly different from its heterozygote (odds ratio [OR] = 3.633, 95% confidence interval [CI]: 2.147–6.148). In conclusion, the results suggest that polymorphism of XRCC1 Arg280His was associated with the increased risk of uterine leiomyoma in a Chinese population.     

Polymorphism of DNA repair gene XRCC1 and hepatitis-related hepatocellular carcinoma risk in Indian population

Background Hepatocellular carcinoma (HCC) is one of the life-threatening malignancies worldwide with hepatitis B and C virus infection as the major risk factor. The risk of HCC might also increase because of the hereditary genetic defects in DNA repair genes. In this regard, X-ray cross-complementing group 1 gene (XRCC1) is a major DNA repair gene involved in base excision repair (BER). Aim The present study was designed with an aim to find out any possible association between XRCC1 (codons 194, 280, and 399) polymorphisms and the risk of developing hepatitis virus-related HCC in Indian population. Methods A total of 407 subjects comprising (170 controls, 174 chronic viral hepatitis, and 63 HCC subjects) were included in the study. PCR–RFLP was used for the genotyping of the three codons of XRCC1. Results The study revealed that two genotypes Arg194Trp and Arg280His increased the risk of HCC by 2.27- (95% CI = 1.01–5.08; P < 0.001) and 4.95-folds (95% CI = 2.48–9.89; P < 0.001), respectively. Interestingly, the risk for HCC was further enhanced by 35.96 (95% CI = 11.64–110.91; P < 0.001) and 5.28 times (95% CI = 2.81–9.09; P < 0.001) when the genotype Arg280His was found in association with Arg194Trp and Arg399Gln, respectively. Conclusion These preliminary results suggest a positive association of XRCC1 genotypes and risk of hepatitis virus-related HCC in India.     

An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.     

Solution structure of the single-strand break repair protein XRCC1 N-terminal domain.

XRCC1 functions in the repair of single-strand DNA breaks in mammalian cells and forms a repair complex with beta-Pol, ligase III and PARP. Here we describe the NMR solution structure of the XRCC1 N-terminal domain (XRCC1 NTD). The structural core is a beta-sandwich with beta-strands connected by loops, three helices and two short two-stranded beta-sheets at each connection side. We show, for the first time, that the XRCC1 NTD specifically binds single-strand break DNA (gapped and nicked). We also show that the XRCC1 NTD binds a gapped DNA-beta-Pol complex. The DNA binding and beta-Pol binding surfaces were mapped by NMR and found to be well suited for interaction with single-strand gap DNA containing a 90 degrees bend, and for simultaneously making contacts with the palm-thumb of beta-Pol in a ternary complex. The findings suggest a mechanism for preferential binding of the XRCC1 NTD to flexible single-strand break DNA.     

Effects on sister chromatid exchange frequency of polymorphisms in DNA repair gene XRCC1 in smokers

The association between metabolic polymorphisms and cigarette smoking-induced cancers has been documented. However, the role of DNA repair polymorphism in carcinogenesis is less clear. To investigate if the polymorphisms of metabolic traits and DNA repair modulate smoking-related DNA damage, we used sister chromatid exchange (SCE) as a marker of genetic damage to explore the relationship of microsomal epoxide hydrolase (mEH), glutathione S-transferase M1 (GSTM1), and X-ray cross-complementing group 1 (XRCC1) and cigarette smoking-induced SCE. Sixty-one workers without significant exposure to mutagens were recruited. Questionnaires were completed to obtain detailed occupational, smoking, and medical histories. SCE frequency in peripheral lymphocytes was determined using a standard cytogenetic assay and GSTM1, mEH (exons 3 and 4), XRCC1 (codon 399) genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP). Smokers had higher SCE frequency than non-smokers (8.4 versus 7.1, P<0.05). Among workers who had smoked equal to or greater than 10 cigarettes each day, those with XRCC1 Arg/Gln+Gln/Gln had higher SCE frequency than those with XRCC1 Arg/Arg after adjusting for potential confounders (9.0 versus 7.9, P<0.05). The interaction of XRCC1 and cigarettes smoked per day on SCE frequency was also observed (P=0.02). There was no significant interaction between cigarettes smoked per day with GSTM1 and mEH on SCE frequency. Our results support previous epidemiological studies that XRCC1 may play a role in cigarette smoking-induced lung cancer.     

Influence of DNA repair gene polymorphisms of hOGG1, XRCC1, XRCC3, ERCC2 and the folate metabolism gene MTHFR on chromosomal aberration frequencies

We have studied the effect of genetic polymorphisms in the DNA repair genes hOGG1, XRCC1, XRCC3, ERCC2 and the MTHFR gene in the folate metabolism on the frequencies of cells with chromosomal aberrations (CA), chromosome-type aberrations (CSA), chromatid-type aberrations (CTA), chromatid breaks (CTB) and chromatid gaps (CTG) scored in peripheral blood lymphocytes from 651 Norwegian subjects of Caucasian descendant. DNA was extracted from fixed cell suspensions. The log-linear Poisson regression model was used for the combined data which included age, smoking, occupational exposure and genotype for 449 subjects.

Our results suggest that individuals carrying the hOGG1 326Cys or the XRCC1 399Gln allele have an increased risk of chromosomal damage, while individuals carrying the XRCC1 194Trp or the ERCC2 751Gln allele have a reduced risk regardless of smoking habits and age.

Individuals carrying the XRCC1 280His allele had an increased risk of CSA which was only apparent in non-smokers. This was independent of age.

A protective effect of the XRCC3 241Met allele was only found in the older age group in non-smokers for CA, CSA and CTA, and in smokers for CSA. In the youngest age group, the opposite effect was found, with an increased risk for CA, CTA and CTG in smokers. Carrying the MTHFR 222Val allele gave an increased risk for chromosome and chromatid-type aberrations for both non-smokers and smokers, especially for individuals in the older age group, and with variable results in the youngest age group. The variables included in the different regression models accounted, however, for only 4–10% of the variation. The frequency ratio for CTG was significantly higher than for CTA and CTB for only 7 of the 43 comparisons performed. Some of the gap frequencies diverge from the trend in the CA, CSA, CTA and CTB results.     

Direct interaction between XRCC1 and UNG2 facilitates rapid repair of uracil in DNA by XRCC1 complexes

Uracil-DNA glycosylase, UNG2, interacts with PCNA and initiates post-replicative base excision repair (BER) of uracil in DNA. The DNA repair protein XRCC1 also co-localizes and physically interacts with PCNA. However, little is known about whether UNG2 and XRCC1 directly interact and participate in a same complex for repair of uracil in replication foci. Here, we examine localization pattern of these proteins in live and fixed cells and show that UNG2 and XRCC1 are likely in a common complex in replication foci. Using pull-down experiments we demonstrate that UNG2 directly interacts with the nuclear localization signal-region (NLS) of XRCC1. Western blot and functional analysis of immunoprecipitates from whole cell extracts prepared from S-phase enriched cells demonstrate the presence of XRCC1 complexes that contain UNG2 in addition to separate XRCC1 and UNG2 associated complexes with distinct repair features. XRCC1 complexes performed complete repair of uracil with higher efficacy than UNG2 complexes. Based on these results, we propose a model for a functional role of XRCC1 in replication associated BER of uracil.     

E2F1 is involved in DNA single-strand break repair through cell-cycle-dependent upregulation of XRCC1 expression

The X-ray repair cross complementing group 1 (XRCC1) protein is involved in DNA base excision repair and its expression varies during the cell cycle. Although studies have demonstrated that rapid XRCC1-dependent single-strand break repair (SSBR) takes place specifically during S/G(2) phases, it remains unclear how it is regulated during the cell cycle. We found that XRCC1 is a direct regulatory target of E2F1 and further investigated the role of XRCC1 in DNA repair during the cell cycle. Saos2 primary osteosarcoma cells stably transfected with inducible E2F1-wt or mutant E2F1-132E were treated with hydroxurea (HU) for 36h and were subsequently withdrawn HU for 2-24h to test whether cell-cycle-dependent DNA SSBR requires E2F1-mediated upregulation of XRCC1. We found that SSBR activity, as determined using a qPCR-base method, was correlated with E2F1 levels at different phases of the cell cycle. XRCC1-positive (AA8) and negative (EM9) CHO cells were used to demonstrate that the alterations in SSBR were mediated by XRCC1. The results indicate that E2F1-mediated regulation of XRCC1 is required for cell-cycle-dependent SSBR predominantly in G(1)/S phases. Our observations have provided new mechanistic insight for understanding the role of E2F1 in the maintenance of genomic stability and cell survival during the cell cycle. The regulation of XRCC1 by E2F1 during cell-cycle-dependent SSBR might be an important aspect for practical consideration for resolving the problem of drug resistance in tumor chemotherapies.     

Variation in DNA repair gene XRCC3 affects susceptibility to astrocytomas and glioblastomas

The gene XRCC3 (X-ray cross complementing group 3) has the task of repairing damage that occurs when there is recombination between homologous chromosomes. Repair of recombination between homologous chromosomes plays an important role in maintaining genome integrity, although it is known that double-strand breaks are the main inducers of chromosomal aberrations. Changes in the XRCC3 protein lead to an increase in errors in chromosome segregation due to defects in centrosomes, resulting in aneuploidy and other chromosomal aberrations, such as small increases in telomeres. We examined XRCC3 Thr241Met polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. The individuals of the control group (N = 100) were selected from the general population of the S?o Paulo State. Odds ratio and 95%CI were calculated using a logistic regression model. Patients who had the allele Met of the XRCC3 Thr241Met polymorphism had a significantly increased risk of tumor development (odds ratio = 3.13; 95% confidence interval = 1.50-6.50). There were no significant differences in overall survival of patients. We suggest that XRCC3 Thr241Met polymorphism is involved in susceptibility for developing astrocytomas and glioblastomas.     

The ataxia-oculomotor apraxia 1 gene product has a role distinct from ATM and interacts with the DNA strand break repair proteins XRCC1 and XRCC4

Ataxia-oculomotor apraxia 1 (AOA1) is an autosomal recessive neurodegenerative disease that is reminiscent of ataxia-telangiectasia (A-T). AOA1 is caused by mutations in the gene encoding aprataxin, a protein whose physiological function is currently unknown. We report here that, in contrast to A-T, AOA1 cell lines exhibit neither radioresistant DNA synthesis nor a reduced ability to phosphorylate downstream targets of ATM following DNA damage, suggesting that AOA1 lacks the cell cycle checkpoint defects that are characteristic of A-T. In addition, AOA1 primary fibroblasts exhibit only mild sensitivity to ionising radiation, hydrogen peroxide, and methyl methanesulphonate (MMS). Strikingly, however, aprataxin physically interacts in vitro and in vivo with the DNA strand break repair proteins XRCC1 and XRCC4. Aprataxin possesses a divergent forkhead associated (FHA) domain that closely resembles the FHA domain present in polynucleotide kinase, and appears to mediate the interactions with CK2-phosphorylated XRCC1 and XRCC4 through this domain. Aprataxin is therefore physically associated with both the DNA single-strand and double-strand break repair machinery, raising the possibility that AOA1 is a novel DNA damage response-defective disease.     

Polymorphisms of the DNA repair gene XRCC1 and the frequency of somatic mutations at the glycophorin A locus in newborns

Essential oils extracted from the three medicinal plants; Helichrysum italicum, Ledum groenlandicum and Ravensara aromatica, together with their mixture were tested for their genotoxic and antigenotoxic activities against urethane, a well-known promutagen. We have adopted the somatic mutations and recombination test (SMART) in the wings of Drosophila melanogaster. Three days old larvae, trans-heterozygous for two genetic markers mwh and flr, were treated by essential oil and/or urethane. A negative control corresponding to solvent was also used. Our results do not show any significant effect of the oils tested but they reduce the mutation ratio resulting from urethane. The mixture of the three oils at equal volume seems to be the most effective. The antimutagenic effect of these oils could be explained by the interaction of their constituents with cytochrome P-450 activation system leading to a reduction of the formation of the active metabolite. The effect could also be attributed to certain molecules that are involved in these oils.     

XRCC1 prevents toxic PARP1 trapping during DNA base excision repair

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