Polymorphisms of DNA repair genes ERCC2 and XRCC1 in populations of Russia

We have conducted a comparative study of allele frequencies of single nucleotide polymorphisms (SNPs) rs1799793 and rs13181 of the ERCC2 gene as well as rs1799782 and rs25487 of the XRCC1 gene in population samples from European regions of Russia as well as in populations of Izhemsk and Priluzsk Komi and Yakuts. Significant differences in the distribution of polymorphic variants of the ERCC2 gene were demonstrated between populations of Yakuts and populations of Russians and Komi. In case of XRCC1 gene Izhemsk Komi population exhibited dissimilar allele frequencies compared to other populations.     

DNA repair genes hOGG1, XRCC1 and ERCC2 polymorphisms and their molecular mapping in breast cancer patients from India

Molecular Biology Reports - Identification of modifier genes predisposing to breast cancer (BC) phenotype remains a significant challenge and varies with ethnicity. The genetic variability observed...     

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.     

The polymorphism of DNA repair genes XPD,XRCC1, OGG1, and ERCC6, life expectancy,and the inclination to smoke

The polymorphism of excision repair genes XPD Asp312Asn, XRCC1 Arg399Gln, OGG1 Ser326Cys, and ERCC6 Met1097Val was analyzed by PCR-RFLP in 370 representatives of the Belarusian population of average, old, and elderly ages. Correlation analysis showed that the frequencies of wild-type homozygous combinations significantly increase with age in the group of subjects over 70 years old in the case of the interaction of two genes, XPD 312 and XRCC1 399, or three genes, XPD 312, XRCC1 399, and ERCC6 1097. In a subgroup of the long-lived, this relationship is manifested in case of a pairwise interaction of gene XPD 312 with XRCC1 399 or ERCC6 1097, as well as an interaction of three genes, XPD 312, XRCC1 399, and ERCC6 1097. The data suggest that the optimum activity of repair processes may favor longevity. It is shown that the frequency of the Asp/Asp genotype is reduced, and the frequency of the Asn allele of the XPD 312 gene is increased in the subgroup of smokers as compared with nonsmokers, which apparently indicates an association of this gene polymorphism with an inclination to smoke. The problem requires further study.     

Domain mapping of the DNA binding, endonuclease, and ERCC1 binding properties of the human DNA repair protein XPF.

During nucleotide excision repair, one of the two incisions necessary for removal of a broad spectrum of DNA adducts is made by the human XPF/ERCC1 protein complex. To characterize the biochemical function of XPF, we have expressed and purified the independent 104 kDa recombinant XPF protein from E. coli and determined that it is an endonuclease and can bind DNA in the absence of the ERCC1 subunit. Endonuclease activity was also identified in a stable 70 kDa proteolysis fragment of XPF obtained during protein expression, indicating an N-terminal catalytic domain. Sequence homology and secondary structure predictions indicated a second functional domain at the C-terminus of XPF. To investigate the significance of the two predicted domains, a series of XPF deletion fragments spanning the entire protein were designed and examined for DNA binding, endonuclease activity, and ERCC1 subunit binding. Our results indicate that the N-terminal 378 amino acids of XPF are capable of binding and hydrolyzing DNA, while the C-terminal 214 residues are capable of binding specifically to ERCC1. We propose that the N-terminal domain of XPF contributes to the junction-specific endonuclease activity observed during DNA repair and recombination events. In addition, evidence presented here suggests that the C-terminal domain of XPF is responsible for XPF/ERCC1 complex formation. A working model for the XPF protein is presented illustrating the function of XPF in the nucleotide excision pathway and depicting the two functional domains interacting with DNA and ERCC1.     

DNA repair gets physical: mapping an XPA-binding site on ERCC1

Two recent reports provide new physical information on how the XPA protein recruits the ERCC1-XPF heterodimer to the site of damage during the process of mammalian nucleotide excision repair (NER). Using chemical shift perturbation NMR experiments, the contact sites between a central fragment of ERCC1 and an XPA fragment have been mapped. While both studies agree with regard to the XPA-binding site, they differ on whether the ERCC1-XPA complex can simultaneously bind DNA. These studies have important implications for both the molecular process and the design of potential inhibitors of NER.     

The XRCC2 and XRCC3 repair genes are required for chromosome stability in mammalian cells.

The irs1 and irs1SF hamster cell lines are mutated for the XRCC2 and XRCC3 genes, respectively. Both show heightened sensitivity to ionizing radiation and particularly to the DNA cross-linking chemical mitomycin C (MMC). Frequencies of spontaneous chromosomal aberration have previously been reported to be higher in these two cell lines than in parental, wild-type cell lines. Microcell-mediated chromosome transfer was used to introduce complementing or non-complementing human chromosomes into each cell line. irs1 cells received human chromosome 7 (which contains the human XRCC2 gene) or, as a control, human chromosome 4. irs1SF cells received human chromosome 14 (which contains the XRCC3 gene) or human chromosome 7. For each set of hybrid cell lines, clones carrying the complementing human chromosome recovered MMC resistance to near-wild-type levels, while control clones carrying noncomplementing chromosomes remained sensitive to MMC. Fluorescence in situ hybridization with a human-specific probe revealed that the human chromosome in complemented clones remained intact in almost all cells even after extended passage. However, the human chromosome in noncomplemented clones frequently underwent chromosome rearrangements including breaks, deletions, and translocations. Chromosome aberrations accumulated slowly in the noncomplemented clones over subsequent passages, with some particular deletions and unbalanced translocations persistently transmitted throughout individual subclones. Our results indicate that the XRCC2 and XRCC3 genes, which are now considered members of the RAD51 gene family, play essential roles in maintaining chromosome stability during cell division. This may reflect roles in DNA repair, possibly via homologous recombination.     

The polymorphism of DNA repair genes XRCC1, XRCC3 and the level of chromosomal aberrations in the Uranium workers

Association between the polymorphism of DNA repair genes XRCC1 Arg399ln and XRCC3 Thr241Met and the frequency of chromosomal aberrations in the uranium workers was studied. The Gln/Gln genotype of gene XRCC1 was associated with a significant increase in the number of chromosomal aberrations as compared to the corresponding homozygous wild type Arg/Arg (p < 0.05). The frequency of chromosomal aberrations in heterozygous carriers of the XRCC3gene Thr/Met was lower than in the homozygous carriers of the wild type Thr/Thr (p < 0.001).     

Epidermal growth factor and ionizing radiation up-regulate the DNA repair genes XRCC1 and ERCC1 in DU145 and LNCaP prostate carcinoma through MAPK signaling

This work examined the importance of radiation-induced and ligand-induced EGFR-ERK signaling for the regulation of DNA repair proteins XRCC1 and ERCC1 in prostate carcinoma cells, DU145 (TP53(mut)), displaying EGFR-TGFA-dependent autocrine growth and high MAPK (ERK1/2) activity, and LNCaP (TP53(wt)) cells expressing low constitutive levels of ERK1/2 activity. Using quantitative RT-PCR and Western analyses, we determined that ionizing radiation activated the DNA repair genes XRCC1 and ERCC1 in an ERK1/2-dependent fashion for each cell line. After irradiation, a rapid increase followed by a decrease in ERK1/2 activity preceded the increase in XRCC1/ERCC1 expression in DU145 cells, while only the rapid decrease in ERK1/2 preceded the increase in XRCC1/ERCC1 expression in LNCaP cells. Administration of EGF, however, markedly increased the up-regulation of phospho-ERK, ERCC1 and XRCC1 in both cell lines. Although the EGFR inhibitor tyrphostin (AG-1478) and the MEK inhibitor PD90859 both attenuated EGF-induced levels of the ERCC1 and XRCC1 protein, PD98059 blocked the induction of ERCC1 and XRCC1 by radiation more effectively in both cell lines. Inhibition of ERK at a level that reduced the up-regulation of DNA repair led to the persistence of apurinic/apyrimidinic (AP) sites of DNA damage and increased cell killing. Taken together, these data imply a complex control of DNA repair activation that may be more generally dependent on MAPK (ERK1/2) signaling than was previously noted. These data provide novel insights into the capacity of the EGFR-ERK signaling to modulate DNA repair in cancer cells and into the functional significance of this signaling.     

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.     

Assignment of a human DNA double-strand break repair gene (XRCC5) to chromosome 2.

The Chinese hamster ovary (CHO-K1) cell mutant XRS-6 is defective in rejoining of DNA double-strand breaks and is hypersensitive to X-rays, gamma-rays, and bleomycin. Radiation resistance or sensitivity of somatic cell hybrids constructed from the fusion of XRS-6 cells with primary human fibroblasts strongly correlated with the retention of human chromosome 2 isozyme and molecular markers. Discordancies between some chromosome 2 markers and the radiation resistance phenotype in some of the hybrid cells suggested the location of the X-ray repair cross complementing 5 (XRCC5) gene on the p arm of chromosome 2. Introduction of human chromosome 2 by microcell-mediated chromosome transfer into the radiation-sensitive XRS-6 cells resulted in hybrid cells in which the radiation sensitivity was complemented. The chromosome 2p origin of the complementing human DNA in the microcell hybrids was supported by fluorescent in situ hybridization analysis of human metaphases using human DNA amplified from the hybrids by inter-Alu-PCR as chromosome-painting probes. XRCC5 is therefore provisionally assigned to human chromosome 2p.     

Linkage assignment of a DNA sequence (ERCC2L1) homologous to a human DNA repair gene in Xiphophorus fishes: implications for the evolutionary derivation of human chromosome 19.

Fish gene mapping studies have identified several syntenic groups showing conservation over more than 400 million years of vertebrate evolution. In particular, Xiphophorus linkage group IV has been identified as a homolog of human chromosomes 15 and 19. During mammalian evolution, loci coding for glucosephosphate isomerase, peptidase D, muscle creatine kinase, and several DNA repair genes (ERCC1, ERCC2, and XRCC1) appear as a conserved syntenic group on human chromosome 19. When X. clemenciae and X. milleri PstI endonuclease-digested genomic DNA was used in Southern analysis with a human ERCC2 DNA repair gene probe, a strongly cross-hybridizing restriction fragment length polymorphism was observed. Backcrosses to X. clemenciae from X. milleri x X. clemenciae F1 hybrids allowed tests for linkage of the ERCC2-like polymorphism to markers covering a large proportion of the genome. Statistically significant evidence for linkage was found only for ERCC2L1 and CKM (muscle creatine kinase), with a total of 41 parents and 2 recombinants (4.7% recombination, chi 2 = 35.37, P less than 0.001); no evidence for linkage to GPI and PEPD in linkage group IV was detected. The human chromosome 19 synteny of ERCC2 and CKM thus appears to be conserved in Xiphophorus, while other genes located nearby on human chromosome 19 are in a separate linkage group in this fish. If Xiphophorus gene arrangements prove to be primitive, human chromosome 19 may have arisen from chromosome fusion or translocation events at some point since divergence of mammals and fishes from a common ancestor.     

Laryngeal cancer risk and common single nucleotide polymorphisms in nucleotide excision repair pathway genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5 and XPA

Because the molecular mechanisms underlying the development of laryngeal cancer are not well understood, we conducted a case–control study to determine the association between eight common SNPs in NER pathway genes and risk of laryngeal cancer, and the association between genetic polymorphisms and environmental factors. A 1:1 matched case–control study of 176 cases and 176 controls was conducted. Laryngeal cancer cases were more likely to smoke and drink (all P values < 0.05). Subjects with the ERCC1 rs11615 CC genotype and C allele had an increased risk of laryngeal cancer. Similarly, individuals with the ERCC5 rs17655 GG genotype and G allele had an increased risk of laryngeal cancer. Gene–gene interaction analysis showed that subjects carrying ERCC1 rs11615 C allele and XPG/ERCC5 rs17655 G allele had a greatly increased risk of breast cancer. Stratified analysis revealed that the interaction between polymorphisms of ERCC1 rs11615 and ERCC5 rs17655 and smoking on cancer risk was statistically significant, and ERCC1 rs11615 polymorphisms also had a significant interaction with drinking habit. In conclusion, our study suggests that ERCC1 rs11615 and ERCC5 rs17655 polymorphisms are associated with increased risk of laryngeal cancer, and that they confer more risk among smokers and drinkers.     

Mammalian recombination-repair genes XRCC2 and XRCC3 promote correct chromosome segregation

Growth and development are dependent on the faithful duplication of cells. Duplication requires accurate genome replication, the repair of any DNA damage, and the precise segregation of chromosomes at mitosis; molecular checkpoints ensure the proper progression and fidelity of each stage. Loss of any of these highly conserved functions may result in genetic instability and proneness to cancer. Here we show that highly significant increases in chromosome missegregation occur in cell lines lacking the RAD51-like genes XRCC2 and XRCC3. This increased missegregation is associated with fragmentation of the centrosome, a component of the mitotic spindle, and not with loss of the spindle checkpoint. Our results show that unresolved DNA damage triggers this instability, and that XRCC2 and XRCC3 are potential tumour-suppressor genes in mammals.     

Breast cancer risk and common single nucleotide polymorphisms in homologous recombination DNA repair pathway genes XRCC2, XRCC3, NBS1 and RAD51

The possible role for DNA repair deficiencies in cancer development, namely in breast cancer has been the subject of increasing interest since it has been reported that breast cancer patients might be deficient in the repair of DNA damage. Exposure to ionizing radiation has been pointed out as a risk factor for breast cancer, and the type of DNA lesions induced by this carcinogen can be repaired by homologous recombination DNA repair (HRR) pathway. To evaluate the potential modifying role of some single nucleotide polymorphisms (SNP) in HRR involved genes on the individual susceptibility to breast cancer we carried out a hospital based case–control study in a Caucasian Portuguese population (289 histological confirmed breast cancer patients and 548 control individuals). We genotyped 4 SNPs in 4 different HRR pathway genes, XRCC2 (Ex3 + 442G > A, R188H, rs3218536), XRCC3 (Ex8-5C > T, T241M, rs861539), NBS1 (Ex5-32C > G, E185Q, rs1805794) and RAD51 5′UTR (Ex1-59G > T, rs1801321), tagging 41 SNPs in these genes. The frequency of the different polymorphisms in the Portuguese control population is similar to the ones reported for other Caucasian populations, and the deviation of the Hardy–Weinberg equilibrium was only observed for the XRCC2 (Ex3 + 442G > A, R188H, rs3218536) polymorphism in the control population. The results obtained, after logistic regression analysis, did not reveal a major role of these polymorphisms on breast cancer susceptibility. However, when the population was stratified according to breast feeding (women that breast fed and women that never breast fed) it is observed, in women that never breast fed, that the heterozygous individuals for the XRCC2 (Ex3 + 442G > A, R188H, rs3218536) polymorphism have a decreased risk for breast cancer [adjusted OR = 0.45; 95% CI = 0.22–0.92] (P = 0.03). Additionally, after stratification according to menopausal status, our results suggest that post-menopausal women carrying at least one variant allele for the XRCC3 (Ex8-5C > T, T241M, rs861539) polymorphism have a lower risk for breast cancer [adjusted OR = 0.67; 95% CI, 0.47–0.94] (P = 0.03). Most of the studies suggest that breastfeeding may be responsible for 2/3 of the estimate reduction of breast cancer. The longer the duration of breastfeeding the lower the potential risk associated with breast cancer. Therefore, in our study the potential protective role of the variant allele of XRCC2 (Ex3 + 442G > A, R188H, rs3218536), in never breast fed women, might be related with a more efficient DNA repair activity.