XRCC2 Human

What is the XRCC2 gene and what is its role in DNA repair?

XRCC2 is a novel member of the RecA/RAD51 recombination repair gene family that plays a crucial role in the homologous recombination repair of DNA double-strand breaks. The gene encodes a protein that functions in high-fidelity DNA repair processes in mammals. XRCC2 is structurally distinct from other family members but retains highly conserved nucleotide binding domains characteristic of the RecA/RAD51 family, including the conserved GxxxxGKT structure in the first domain and a characteristic hydrophobic β-sheet in the second domain . The protein is smaller than other members of this family, which may reflect its specialized role in DNA repair mechanisms .

Unlike RAD51 which functions in both meiosis and mitosis, XRCC2 expression is very low in somatic tissues but elevated in mouse testis, suggesting an additional role in meiosis beyond its somatic DNA repair functions . Cell lines with XRCC2 mutations (like irs1) show extreme sensitivity to DNA cross-linking agents and significant genetic instability, confirming its crucial role in maintaining genomic integrity .

What is the genomic structure of the human XRCC2 gene?

The human XRCC2 gene is located on chromosome 7q36.1. The gene is divided into three exons with intron-exon boundaries conforming to the consensus AG at the 3' splice site and GT at the 5' splice site . The total length of human XRCC2 cDNA sequence is approximately 1580 bp, with a potential start codon at position 83 that predicts an open reading frame of 840 bp .

The gene contains a CpG island in close proximity to the 5' end, which is typical of housekeeping genes . This CpG island structure is conserved between human and mouse genomes, being located at the same relative position to the 5' end of the XRCC2 gene in both species . The 3' untranslated region (UTR) of the human gene contains an Alu repeat sequence and has one of the less common variants of the polyadenylation signal sequence (ATTAAA) .

What experimental models are available for studying XRCC2 function?

Several experimental models have been developed to study XRCC2 function:

• Cell line models: The irs1 cell line derived from V79-4 Chinese hamster cells contains a mutation in the XRCC2 gene and displays extreme sensitivity to DNA cross-linking agents and genetic instability . This cell line has been fundamental for characterizing XRCC2 function.

• Complementation systems: The GT621-1 cell line (irs1 transfected with the XRCC2 gene) shows full or partial complementation of radiosensitivity and other defects, making it useful for studying XRCC2 rescue effects .

• Knockout mouse models: Targeted mutagenesis of mouse Xrcc2 has been achieved by disrupting the gene in 129/Sv/Ev embryonic stem cells. This disruption involves introducing a neomycin-resistance cassette that deletes exon III (86% of the coding region), which encodes the essential nucleotide binding motifs .

• Transfection models: Human cDNA for XRCC2 in expression vectors can be transfected into XRCC2-deficient cells to study complementation. Successful transfection has been demonstrated using hygromycin resistance selection, with pools of approximately 100 transfected lines tested for resistance to DNA-damaging agents like mitomycin C .

How can researchers assess XRCC2-dependent DNA repair activity?

XRCC2-dependent DNA repair activity can be assessed through several experimental approaches:

• Sensitivity to DNA cross-linking agents: Exposing cell lines to mitomycin C and measuring survival rates provides a direct measure of XRCC2 functionality. Wild-type cells, XRCC2-mutant cells, and complemented cells can be resuspended in different concentrations of mitomycin C to establish dose-response curves .

• Chromosomal aberration analysis: Cycling cultures of cells can be examined for the frequency of chromosomal aberrations by scoring Giemsa-stained metaphase spreads according to standard criteria. This provides a measure of genetic instability resulting from XRCC2 deficiency .

• Chromatid break analysis: The color-switch ratio (CSR) in harlequin-stained cells can be analyzed by labeling cells with BrdU through two cell cycles, irradiating them, and sampling 1.5h after exposure. Metaphase spreads can then be analyzed for chromatid break frequency and frequencies of color-switch and non-color-switch breaks .

How do XRCC2 polymorphisms influence cancer susceptibility?

XRCC2 polymorphisms have been significantly associated with breast cancer risk and survival. The most significant association with breast cancer risk has been found with the XRCC2 rs3218408 SNP (MAF=0.23) . This SNP demonstrated an odds ratio (OR) of 1.64 (95% CI: 1.25-2.16) in a two-site analysis of the Sheffield Breast Cancer Study (SBCS) and Utah Breast Cancer Study (UBCS), and a meta-OR of 1.33 (95% CI: 1.12-1.57) when all published data were included .

This particular SNP may mark a rare risk haplotype carried by approximately 2 in 1000 individuals in the control population . The association follows a recessive model, suggesting that individuals homozygous for the risk allele have a significantly increased susceptibility to breast cancer.

What is the relationship between XRCC2 variants and cancer prognosis?

The XRCC2 coding R188H SNP (rs3218536, MAF=0.08) has been significantly associated with poor survival in breast cancer patients . In multivariate analysis, this SNP demonstrated an increased per-allele hazard ratio (HR) of 1.58 (95% CI: 1.01-2.49) .

This survival effect remained evident in a pooled meta-analysis of 8,781 breast cancer patients from the Breast Cancer Association Consortium (BCAC), showing an HR of 1.19 (95% CI: 1.05-1.36, p=0.01) . This suggests that the R188H variant may influence DNA repair capacity in a way that affects tumor progression or response to treatment, ultimately impacting patient outcomes.

The table below summarizes key XRCC2 polymorphisms associated with breast cancer:

How does XRCC2 contribute to homologous recombination at the molecular level?

XRCC2 plays a crucial role in homologous recombination repair by influencing the control of intra-versus interchromatid rearrangements that lead to chromatid breaks in G2 cells . Studies of Chinese hamster irs1 cells (with XRCC2 mutation) demonstrate that XRCC2 affects the color-switch ratio (CSR), which is a measure of the relative frequency of inter- versus intrachromatid exchanges at break points .

The CSR was significantly higher in irs1 cells (13.9%) compared to the parental V79-4 wild-type cells (7.5%) or irs2 cells with XRCC8 mutation (4.9%) . When irs1 cells were transfected with the XRCC2 gene (GT621-1 cells), they showed partial but significant complementation with respect to CSR (9.2%) .

These findings indicate that XRCC2 influences which chromatid is used as a template for repair, potentially by facilitating the search for homology or by promoting strand invasion during the homologous recombination process. The higher CSR in XRCC2-deficient cells suggests that without functional XRCC2, cells tend to favor interchromatid exchanges over intrachromatid exchanges during repair .

What role does XRCC2 play in embryonic development?

XRCC2 is essential for embryonic neurogenesis and viability in mice . Targeted disruption of Xrcc2 in mice by deleting exon III, which encodes the nucleotide binding motifs essential for activity, demonstrates its crucial developmental functions .

The deleted region in knockout models encodes not only the nucleotide binding motifs conserved in the RecA/Rad51 gene family but also the entire 3' end of the gene including poly(A) signal sites . This disruption results in a complete loss of functional Xrcc2 protein.

Homozygous Xrcc2-knockout embryos show severe developmental abnormalities, particularly in the nervous system, suggesting that proper DNA repair through homologous recombination is essential for normal neuronal development. The embryonic lethality observed in these models underscores the fundamental importance of XRCC2 in mammalian development .

What techniques are most effective for studying XRCC2 genetic variants in human populations?

Several methodologies have proven effective for studying XRCC2 genetic variants in population studies:

• Tagging SNP approach: Studies have successfully employed a tagging SNP approach to capture common genetic variation across the XRCC2 gene. For example, the Sheffield Breast Cancer Study genotyped 12 XRCC2 tagging SNPs in 1,131 breast cancer cases and 1,148 controls to examine their associations with breast cancer risk and survival .

• Association analysis: Case-control studies examining XRCC2 variants typically estimate odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) to quantify associations with disease risk . For survival analysis, hazard ratios (HRs) are calculated using Cox proportional hazards models, with adjustment for relevant covariates .

• Meta-analysis: Combining data from multiple studies enhances statistical power and provides more reliable estimates of genetic effects. The meta-analysis approach has been successfully applied to XRCC2 variants, as demonstrated by the pooled analysis of 8,781 breast cancer patients from the Breast Cancer Association Consortium .

• Replication in independent cohorts: Initial findings should be validated in independent study populations. For example, positive associations identified in the Sheffield Breast Cancer Study were further investigated in the Utah Breast Cancer Study (860 cases and 869 controls) and then jointly analyzed with published data .

What cell culture systems are optimal for functional studies of XRCC2?

Several cell culture systems have proven valuable for functional studies of XRCC2:

• V79-4 and derivative cell lines: The wild-type Chinese hamster V79-4 cell line and its XRCC2-mutant derivative irs1 provide an excellent system for studying XRCC2 function through comparative analyses . The irs2 cell line with XRCC8 mutation serves as an additional control.

• Complemented cell lines: The GT621-1 cell line (irs1 transfected with XRCC2) demonstrates partial to full complementation of XRCC2-related phenotypes, making it useful for rescue experiments .

• Human cell lines: HeLa cells have been used successfully for cDNA library construction and expression studies of XRCC2 .

• Embryonic stem cells: For developmental studies, 129/Sv/Ev embryonic stem cells have been employed for targeted mutagenesis of Xrcc2, followed by blastocyst injections to generate chimeric offspring .

For optimal results in functional studies, researchers should consider several factors:

• The cell line's endogenous DNA repair capacity

• Background mutation rates

• Transfection efficiency

• Growth characteristics

• Karyotype stability

What are the emerging areas of XRCC2 research with clinical potential?

Several promising research directions for XRCC2 have potential clinical applications:

• Predictive biomarkers: The association of XRCC2 polymorphisms with cancer outcomes suggests potential utility as predictive biomarkers. The R188H variant (rs3218536) in particular shows promise as a prognostic marker for breast cancer survival .

• Synthetic lethality approaches: Given XRCC2's role in homologous recombination repair, targeting synthetic lethal interactions in XRCC2-deficient tumors could provide novel therapeutic strategies, similar to PARP inhibitors in BRCA-deficient cancers.

• Pharmacogenomics: XRCC2 variants may influence response to DNA-damaging chemotherapeutics. Research exploring how XRCC2 polymorphisms affect treatment outcomes could lead to personalized therapy approaches.

• Gene therapy: For conditions associated with XRCC2 deficiency, gene replacement or CRISPR-based approaches might eventually provide therapeutic options.

• Cancer prevention strategies: Understanding how XRCC2 variants modify cancer risk could inform targeted prevention strategies for high-risk individuals.

How can contradictory findings in XRCC2 research be reconciled?

Researchers may encounter contradictory findings in XRCC2 studies for several reasons:

• Population heterogeneity: Different populations may have varying allele frequencies and linkage disequilibrium patterns, affecting study outcomes. Meta-analyses that include diverse populations can help identify consistent effects .

• Gene-environment interactions: XRCC2 variants may have different effects depending on environmental exposures or lifestyle factors that vary between study populations.

• Methodological differences: Variations in study design, genotyping methods, and statistical approaches can lead to apparently contradictory results. Standardized protocols and reporting can address this issue.

• Sample size limitations: Some studies may be underpowered to detect modest genetic effects. Large consortium-based approaches, such as those employed by the Breast Cancer Association Consortium, can overcome this limitation .

• Publication bias: Positive findings are more likely to be published than negative results, potentially skewing the literature. Comprehensive meta-analyses that include unpublished data can help mitigate this bias.

Researchers should carefully consider these factors when interpreting apparently contradictory findings and design studies with sufficient power and methodological rigor to provide definitive answers.