Roles of XRCC2, RAD51B and RAD51D in RAD51-Independent SSA Recombination

The repair of DNA double-strand breaks by recombination is key to the maintenance of genome integrity in all living organisms. Recombination can however generate mutations and chromosomal rearrangements, making the regulation and the choice of specific pathways of great importance. In addition to end-joining through non-homologous recombination pathways, DNA breaks are repaired by two homology-dependent pathways that can be distinguished by their dependence or not on strand invasion catalysed by the RAD51 recombinase. Working with the plant Arabidopsis thaliana, we present here an unexpected role in recombination for the Arabidopsis RAD51 paralogues XRCC2, RAD51B and RAD51D in the RAD51-independent single-strand annealing pathway. The roles of these proteins are seen in spontaneous and in DSB-induced recombination at a tandem direct repeat recombination tester locus, both of which are unaffected by the absence of RAD51. Individual roles of these proteins are suggested by the strikingly different severities of the phenotypes of the individual mutants, with the xrcc2 mutant being the most affected, and this is confirmed by epistasis analyses using multiple knockouts. Notwithstanding their clearly established importance for RAD51-dependent homologous recombination, XRCC2, RAD51B and RAD51D thus also participate in Single-Strand Annealing recombination.     

XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination

Homologous recombinational repair preserves chromosomal integrity by removing double-strand breaks, cross-links, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2/3, Rad51B/C/D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 exists in a single complex with Rad51C. To examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a nonconservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF Chinese hamster ovary cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, whereas ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones. Because of dysfunction of the mutants, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential. We tested in vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C upon co-expression in bacteria, nickel-affinity purification, and Western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, whereas the K113R mutant did not and was predominantly insoluble. The addition of 5 mm ATP but not ADP also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 probably regulates the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis with both processes being essential for the ability of the complex to participate in homologous recombinational repair.     

XRCC2 is a nuclear RAD51-like protein required for damage-dependent RAD51 focus formation without the need for ATP binding

The human XRCC2 gene was recently identified by its ability to complement a hamster cell line, irs1, which is sensitive to DNA-damaging agents and shows genetic instability. The XRCC2 protein is highly conserved in mammalian species and has structural features, including a putative ATP-binding domain (P-loop), consistent with membership of the RecA/RAD51 family of recombination-repair proteins. We show that a hybrid XRCC2-green fluorescent protein, which was found to be functional by complementation, localizes to the nucleus. We have established a functional link between XRCC2 and RAD51 by looking at damage-dependent RAD51 focus formation in the irs1 cell line. Little or no formation of RAD51 foci occurred in irs1. This effect was specific to the loss of XRCC2 because transfection of the gene into irs1 restored normal levels of focus formation. Surprisingly, XRCC2 genes carrying site-specific mutations in P-loop residues were found to be able to complement the XRCC2-deficient irs1 line for a number of different end points. We conclude that XRCC2 is important in the early stages of homologous recombination in mammalian cells to facilitate RAD51-dependent recombination repair but that it does not make use of ATP binding to promote this function.     

RAD51C interacts with RAD51B and is central to a larger protein complex in vivo exclusive of RAD51.

RAD51B and RAD51C are two of five known paralogs of the human RAD51 protein that are thought to function in both homologous recombination and DNA double-strand break repair. This work describes the in vitro and in vivo identification of the RAD51B/RAD51C heterocomplex. The RAD51B/RAD51C heterocomplex was isolated and purified by immunoaffinity chromatography from insect cells co-expressing the recombinant proteins. Moreover, co-immunoprecipitation of the RAD51B and RAD51C proteins from HeLa, MCF10A, and MCF7 cells strongly suggests the existence of an endogenous RAD51B/RAD51C heterocomplex. We extended these observations to examine the interaction between the RAD51B/RAD51C complex and the other RAD51 paralogs. Immunoprecipitation using protein-specific antibodies showed that RAD51C is central to a single large protein complex and/or several smaller complexes with RAD51B, RAD51D, XRCC2, and XRCC3. However, our experiments showed no evidence for the inclusion of RAD51 within these complexes. Further analysis is required to elucidate the function of the RAD51B/RAD51C heterocomplex and its association with the other RAD51 paralogs in the processes of homologous recombination and DNA double-strand break repair.     

The importance of XRCC2 in RAD51-related DNA damage repair

The repair of DNA damage by homologous recombination (HR) is a key pathway for the maintenance of genetic stability in mammalian cells, especially during and following DNA replication. The central HR protein is RAD51, which ensures high fidelity DNA repair by facilitating strand exchange between damaged and undamaged homologous DNA segments. Several RAD51-like proteins, including XRCC2, appear to help with this process, but their roles are not well understood. Here we show that XRCC2 is highly conserved and that most substantial truncations of the protein destroy its ability to function. XRCC2 and its partner protein RAD51L3 are found to interact with RAD51 in the 2-hybrid system, and XRCC2 is shown to be important but not essential for the accumulation of RAD51 at the sites of DNA damage. We visualize the localization of XRCC2 protein at the same sites of DNA damage for the first time using specialized irradiation conditions. Our data indicate that an important function of XRCC2 is to enhance the activity of RAD51, so that the loss of XRCC2 results in a severe delay in the early response of RAD51 to DNA damage.     

RAD51C (RAD51L2) is involved in maintaining centrosome number in mitosis

The RAD51C (RAD51L2) protein is one out of five RAD51 paralogs and forms a complex that includes either XRCC2 or XRCC3. Both of these complexes may have important functions in homologous recombination (HR). Here, we confirm that the frequency of DNA double-strand break (DSB)-induced HR is reduced in the RAD51C deficient cell line CL-V4B, in agreement with a role for RAD51C in HR. We report that mitotic RAD51C deficient CL-V4B cells also have an increased number of centrosomes in mitosis resulting in aberrant mitotic spindles. These data suggest that the RAD51C protein is important in maintaining correct centrosome numbers and that the complexes including RAD51C and XRCC2 or XRCC3 may be of importance in maintaining correct centrosome numbers in mitosis. Increased centrosome numbers following a RAD51C defect indicates that this protein might be important in preventing aneuploidy, suggesting that it could be a potential tumour suppressor in mammals.     

The interaction profile of homologous recombination repair proteins RAD51C,RAD51D and XRCC2 as determined by proteomic analysis

The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co‐precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1‐D SDS‐PAGE of co‐precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co‐precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.     

Functional interaction between the Bloom's syndrome helicase and the RAD51 paralog,RAD51L3 (RAD51D)

Bloom's syndrome (BS) is a genetic disorder associated with short stature, fertility defects, and a predisposition to the development of cancer. BS cells are characterized by genomic instability; in particular, a high rate of reciprocal exchanges between sister-chromatids and homologous chromosomes. The BS gene product, BLM, is a helicase belonging to the highly conserved RecQ family. BLM is known to form a complex with the RAD51 recombinase, and to act upon DNA intermediates that form during homologous recombination, including D-loops and Holliday junctions. Here, we show that BLM also makes a direct physical association with the RAD51L3 protein (also known as RAD51D), a so-called RAD51 paralog that shows limited sequence similarity to RAD51 itself. This interaction is mediated through the N-terminal domain of BLM. To analyze functional interactions between BLM and RAD51L3, we have purified a heteromeric complex comprising RAD51L3 and a second RAD51 paralog, XRCC2. We show that the RAD51L3-XRCC2 complex stimulates BLM to disrupt synthetic 4-way junctions that model the Holliday junction. We also show that a truncated form of BLM, which retains helicase activity but is unable to bind RAD51L3, is not stimulated by the RAD51L3-XRCC2 complex. Our data indicate that the activity of BLM is modulated through an interaction with the RAD51L3-XRCC2 complex, and that this stimulatory effect on BLM is dependent upon a direct physical association between the BLM and RAD51L3 proteins. We propose that BLM co-operates with RAD51 paralogs during the late stages of homologous recombination processes that serve to restore productive DNA replication at sites of damaged or stalled replication forks.     

XRCC3 and RAD51 Expression Are Associated with Clinical Factors in Breast Cancer

Aims

XRCC3 and RAD51 are two important members in homologous recombination repair pathway. This study was performed to detect the expressions of these two molecules in breast cancer and explore their correlations with clinicopathological factors.

Methods and Results

Immunohistochemistry was used to detect protein expressions of XRCC3 and RAD51 in 248 cases of breast cancer tissue and 78 cases of adjacent non-cancerous tissue. Data showed that expressions for both XRCC3 and RAD51 were significantly increased in breast cancer. High XRCC3 expression was associated with large tumor size and positive PR and HER2 status, while high RAD51 expression was associated with axillary lymph node metastasis and positive PR and HER2 status. The result of multivariate analysis demonstrated that HER2, PR and RAD51 were significantly association with XRCC3. And besides XRCC3, axillary lymph node metastasis and PR were significantly correlated with RAD51.

Conclusions

XRCC3 and RAD51 were significantly associated with clinicopathological factors and they might play important roles in the development and progress of breast cancer.     

Association between RAD51, XRCC2 and XRCC3 gene polymorphisms and risk of ovarian cancer: a case control and an in silico study

Molecular Biology Reports - Homologous recombination (HR) is one of the important mechanisms in repairing double-strand breaks to maintain genomic integrity and DNA stability from the cytotoxic...     

RAD51AP1 is a structure-specific DNA binding protein that stimulates joint molecule formation during RAD51-mediated homologous recombination

Homologous recombination is essential for preserving genome integrity. Joining of homologous DNA molecules through strand exchange, a pivotal step in recombination, is mediated by RAD51. Here, we identify RAD51AP1 as a RAD51 accessory protein that specifically stimulates joint molecule formation through the combination of structure-specific DNA binding and physical contact with RAD51. At the cellular level, we show that RAD51AP1 is required to protect cells from the adverse effects of DNA double-strand break-inducing agents. At the biochemical level, we show that RAD51AP1 has a selective affinity for branched-DNA structures that are obligatory intermediates during joint molecule formation. Our results highlight the importance of structural transitions in DNA as control points in recombination. The affinity of RAD51AP1 for the central protein and DNA intermediates of recombination confers on it the ability to control the preservation of genome integrity at a number of critical mechanistic steps.     

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.     

The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family

The genes for two new P-type ATPases, PMR1 and PMR2, have been identified in yeast. A comparison of the deduced sequences of the PMR proteins with other known ion pumps showed that both proteins are very similar to Ca2+ ATPases. PMR1 is identical to SSC1, a gene previously identified by its effect on secretion of some foreign proteins from yeast. Proteins secreted from pmr1 mutants lack the outer chain glycosylation that normally results from passage through the Golgi. Loss of PMR1 function suppresses the lethality of ypt1-1, a mutation that blocks the secretion pathway. These data suggest that PMR1 functions as a Ca2+ pump affecting transit through the secretory pathway.