BRCA1 regulates RAD51 function in response to DNA damage and suppresses spontaneous sister chromatid replication slippage: implications for sister chromatid cohesion, genome stability, and carcinogenesis

The breast/ovarian cancer susceptibility proteins BRCA1 and BRCA2 maintain genome stability, at least in part, through a functional role in DNA damage repair. They both colocalize with RAD51 at sites of DNA damage/replication and activate RAD51-mediated homologous recombination repair of DNA double-strand breaks (DSB). Whereas BRCA2 interacts directly with and regulates RAD51, the role of BRCA1 in this process is unclear. However, BRCA1 may regulate RAD51 in response to DNA damage or through its ability to interact with and regulate MRE11/RAD50/NBS1 (MRN) during the processing of DSBs into single-strand DNA (ssDNA) ends, prerequisite substrates for RAD51, or both. To test these hypotheses, we measured the effect of BRCA1 on the competition between RAD51-mediated homologous recombination (gene conversion and crossover) versus RAD51-independent homologous recombination [single-strand annealing (SSA)] for ssDNA at a site-specific chromosomal DSB within a DNA repeat, a substrate for both homologous recombination pathways. Expression of wild-type BRCA1 in BRCA1-deficient human recombination reporter cell lines promoted both gene conversion and SSA but greatly enhanced gene conversion. In addition, BRCA1 also suppressed both spontaneous gene conversion and deletion events, which can arise from either crossover or sister chromatid replication slippage (SCRS), a RAD51-independent process. BRCA1 does not seem to block crossover. From these results, we conclude that (a) BRCA1 regulates RAD51 function in response to the type of DNA damage and (b) BRCA1 suppresses SCRS, suggesting a role for this protein in sister chromatid cohesion/alignment. Loss of such control in response to estrogen-induced DNA damage after BRCA1 inactivation may be a key initial event that triggers genome instability and carcinogenesis.     

Secondary BRCA1 and BRCA2 alterations and acquired chemoresistance

Tumor suppressor BRCA1 and BRCA2 are frequently mutated in familial breast and ovarian cancer. More than ten percent of women with breast or ovarian cancer carry BRCA1 or BRCA2 (BRCA1/2) mutations. Cancers that arise in mutation carriers have often lost the wild-type allele through somatic alterations during tumor progression. BRCA1/2 play important roles in homologous recombination repair of DNA double-strand breaks. Because of this, BRCA1/2-deficient cancers often have a better response to DNA cross-linking agents such as platinum analogues and to poly(ADP-ribose) polymerase (PARP) inhibitors. However, over time, the majority of these BRCA1/2-deficient cancers become resistant and patients die from refractory diseases. Three recent studies demonstrated that acquired resistance to platinum analogues or PARP inhibitors in tumors carrying frame-shift BRCA1/2 mutations came from restored BRCA1/2 expression and HR function due to secondary intragenic mutations that corrected the open reading frames of mutated BRCA1/2.     

Distinct RAD51 associations with RAD52 and BCCIP in response to DNA damage and replication stress

RAD51 has critical roles in homologous recombination (HR) repair of DNA double-strand breaks (DSB) and restarting stalled or collapsed replication forks. In yeast, Rad51 function is facilitated by Rad52 and other "mediators." Mammalian cells express RAD52, but BRCA2 may have supplanted RAD52 in mediating RAD51 loading onto ssDNA. BCCIP interacts with BRCA2, and both proteins are important for RAD51 focus formation after ionizing radiation and HR repair of DSBs. Nonetheless, mammalian RAD52 shares biochemical activities with yeast Rad52, including RAD51 binding and single-strand annealing, suggesting a conserved role in HR. Because RAD52 and RAD51 associate, and RAD51 and BCCIP associate, we investigated the colocalization of RAD51 with BCCIP and RAD52 in human cells. We found that RAD51 colocalizes with BCCIP early after ionizing radiation, with RAD52 later, and there was little colocalization of BCCIP and RAD52. RAD52 foci are induced to a greater extent by hydroxyurea, which stalls replication forks, than by ionizing radiation. Using fluorescence recovery after photo bleaching, we show that RAD52 mobility is reduced to a greater extent by hydroxyurea than ionizing radiation. However, BCCIP showed no changes in mobility after hydroxyurea or ionizing radiation. We propose that BCCIP-dependent repair of DSBs by HR is an early RAD51 response to ionizing radiation-induced DNA damage, and that RAD52-dependent HR occurs later to restart a subset of blocked or collapsed replication forks. RAD52 and BRCA2 seem to act in parallel pathways, suggesting that targeting RAD52 in BRCA2-deficient tumors may be effective in treating these tumors.     

Checkpoint kinase 2-mediated phosphorylation of BRCA1 regulates the fidelity of nonhomologous end-joining

The tumor suppressor gene BRCA1 maintains genomic integrity by protecting cells from the deleterious effects of DNA double-strand breaks (DSBs). Through its interactions with the checkpoint kinase 2 (Chk2) kinase and Rad51, BRCA1 promotes homologous recombination, which is typically an error-free repair process. In addition, accumulating evidence implicates BRCA1 in the regulation of nonhomologous end-joining (NHEJ), which may involve precise religation of the DSB ends if they are compatible (i.e., error-free repair) or sequence alteration upon rejoining (i.e., error-prone or mutagenic repair). However, the precise role of BRCA1 in regulating these different subtypes of NHEJ is not clear. We provide here the genetic and biochemical evidence to show that BRCA1 promotes error-free rejoining of DSBs in human breast carcinoma cells while suppressing microhomology-mediated error-prone end-joining and restricting sequence deletion at the break junction during repair. The repair spectrum in BRCA1-deficient cells was characterized by an increase in the formation of >2 kb deletions and in the usage of long microhomologies distal to the break site, compared with wild-type (WT) cells. This error-prone repair phenotype could also be revealed by disruption of the Chk2 phosphorylation site of BRCA1, or by expression of a dominant-negative kinase-dead Chk2 mutant in cells with WT BRCA1. We suggest that the differential control of NHEJ subprocesses by BRCA1, in concert with Chk2, reduces the mutagenic potential of NHEJ, thereby contributing to the prevention of familial breast cancers.     

Distinct functions of BRCA1 and BRCA2 in double-strand break repair

Individuals carrying BRCA mutations are predisposed to breast cancer. The BRCA1 and BRCA2 proteins are required for homologous recombination and DNA break repair, leading to the suggestion that they act in concert. However, direct evidence of a stable BRCA1/BRCA2 complex has not been demonstrated. Rather, the two proteins have been found as constituents of discrete, but perhaps nonexclusive complexes that are critical for repair. We discuss the interaction of BRCA1 with the BACH1 and BARD1 proteins, and suggest that the pleiotropic nature of mutations in BRCA1 may be associated with defects in protein--protein interactions. In contrast, the role of BRCA2 in DNA repair may be more defined by its direct interaction with the RAD51 recombinase.     

BRCA2 regulates homologous recombination in response to DNA damage: implications for genome stability and carcinogenesis

BRCA2 has been implicated in the maintenance of genome stability and RAD51-mediated homologous recombination repair of chromosomal double-strand breaks (DSBs), but its role in these processes is unclear. To gain more insight into its role in homologous recombination, we expressed wild-type BRCA2 in the well-characterized BRCA2-deficient human cell line CAPAN-1 containing, as homologous recombination substrates, either direct or inverted repeats of two inactive marker genes. Whereas direct repeats monitor a mixture of RAD51-dependent and RAD51-independent homologous recombination events, inverted repeats distinguish between these events by reporting RAD51-dependent homologous recombination, gene conversion, and crossover events only. At either repeats, BRCA2 decreases the rate and frequency of spontaneous homologous recombination, but following chromosomal DSBs, BRCA2 increases the frequency of homologous recombination. At direct repeats, BRCA2 suppresses both spontaneous gene conversion and deletions, which can arise either from crossover or RAD51-independent sister chromatid replication slippage (SCRS), but following chromosomal DSBs, BRCA2 highly promotes gene conversion with little effect on deletions. At inverted repeats, spontaneous or DSB-induced crossover events were scarce and BRCA2 does not suppress their formation. From these results, we conclude that (i) BRCA2 regulates RAD51 recombination in response to the type of DNA damage and (ii) BRCA2 suppresses SCRS, suggesting a role for BRCA2 in sister chromatids cohesion and/or alignment. Loss of such control in response to estrogen-induced DNA damage after BRCA2 inactivation may be a key initial event triggering genome instability and carcinogenesis.     

Immunohistochemical expression of DNA repair proteins in familial breast cancer differentiate BRCA2-associated tumors.

PURPOSE: Morphologic and immunohistochemical studies of familial breast cancers have identified specific characteristics associated with BRCA1 mutation-associated tumors when compared with BRCA2 and non-BRCA1/2 tumors, but have not identified differences between BRCA2 and non-BRCA1/2 tumors. Because BRCA1 and BRCA2 genes participate in the DNA repair pathway, we have performed an immunohistochemical study with markers related to this pathway to establish the profile of the three groups. MATERIALS AND METHODS: We have studied two tissue microarrays that include 103 familial and 104 sporadic breast tumors, with a panel of DNA repair markers including ATM, CHEK2, RAD51, RAD50, XRCC3, and proliferating cell nuclear antigen. RESULTS: We found more frequent expression of CHEK2 in BRCA1 and BRCA2 tumors than in non-BRCA1/2 and sporadic tumors. We found absence of nuclear expression and presence of cytoplasmic expression of RAD51 in BRCA2 tumors that differentiate them from other familial tumors. We validated these results with a new series of patient cases. The final study with 253 familial patient cases (74 BRCA1, 71 BRCA2, 108 non-BRCA1/2), and 288 sporadic patient cases, has allowed us to confirm our preliminary results. Because BRCA2 tumors present a specific immunohistochemical profile for RAD51 and CHEK2 markers that is different from non-BRCA1/2 tumors, we have built a multivariate model with these markers that distinguish both tumors with an estimated probability of at least 76%. CONCLUSION: Our results suggest that BRCA2 tumors demonstrate more cytoplasmic and less nuclear RAD51 staining, and increased CHEK2 staining. This pattern may distinguish BRCA2 from familial non-BRCA1/2 tumors.     

Nonhomologous end-joining of ionizing radiation-induced DNA double-stranded breaks in human tumor cells deficient in BRCA1 or BRCA2

Mutations in the BRCA1 or BRCA2 genes predispose to a wide spectrum of familial cancers. The functions of the proteins encoded by BRCA1 and BRCA2 remain to be elucidated, but their interaction and colocalization with hRAD51 suggest a role in homologous recombination and DNA double-strand break (DSB) repair. The role of BRCA1 and BRCA2 in the rejoining of ionizing radiation (IR)-induced DNA DSBs, which may represent a step in the overall process of repair, remains uncertain because recent reports provide conflicting results. Because elucidation of the role of these proteins in DNA DSB rejoining is important for their functional characterization, we reexamined this end point in cells with mutations in either BRCA1 or BRCA2. We show that two pancreatic carcinoma cell lines known to have either wild-type (BxPC3) or mutant forms (Capan-1) of BRCA2 rejoin IR-induced DNA DSBs to a similar extent following biphasic kinetics characterized by a fast and a slow component. Importantly, inactivation of DNA-dependent protein kinase (DNA-PK) by wortmannin generates similar shifts from the fast to the slow component of rejoining in BRCA2-proficient and BRCA2-deficient cells. This suggests that the functioning of either the fast, DNA-PK-dependent component or the slow, DNA-PK-independent component of rejoining is not affected by mutations in BRCA2. Also, a human breast cancer cell line with mutated BRCA1 shows normal rejoining of IR-induced DNA DSBs and levels of inhibition by wortmannin commensurate with the degree of DNA-PK inhibition. These observations fail to confirm a direct role for BRCA1 or BRCA2 in the rejoining of IR-induced DSBs in the genome of human tumor cells and, as a result, an involvement in nonhomologous end-joining. They are in line with similar observations with mutants deficient in genes implicated in homologous recombination and support the view that the radiosensitivity to killing of cells deficient in BRCA1 or BRCA2 derives from defects in this repair pathway.     

Distinct functions of BRCA1 and BRCA2 in double-strand break repair

Individuals carrying BRCA mutations are predisposed to breast cancer. The BRCA1 and BRCA2 proteins are required for homologous recombination and DNA break repair, leading to the suggestion that they act in concert. However, direct evidence of a stable BRCA1/BRCA2 complex has not been demonstrated. Rather, the two proteins have been found as constituents of discrete, but perhaps nonexclusive complexes that are critical for repair. We discuss the interaction of BRCA1 with the BACH1 and BARD1 proteins, and suggest that the pleiotropic nature of mutations in BRCA1 may be associated with defects in protein–protein interactions. In contrast, the role of BRCA2 in DNA repair may be more defined by its direct interaction with the RAD51 recombinase.     

Strand invasion involving short tract gene conversion is specifically suppressed in BRCA2-deficient hamster cells

The BRCA2 tumour suppressor protein is involved in maintaining genetic stability through its role in homologous recombination (HR), where it mediates RAD51-dependent strand invasion. Here, we show that BRCA2-defective cells are not completely impaired in HR by strand invasion although the spontaneous HR rate is 10-fold lower than that in wild-type cells. Furthermore, a DNA double-strand break (DSB) triggers HR repair by strand invasion also in BRCA2-defective cells, but less efficiently. Thus, either the strand invasion pathway(s) in which BRCA2 operates is still operative in the absence of a functional BRCA2, albeit at a reduced frequency, or there is a separate pathway for strand invasion still functional in BRCA2-deficient cells. Consistent with the latter hypothesis, we show that HR events occurring in BRCA2-defective cells differ from HR events in wild-type cells. These data suggest that BRCA2-defective hamster cells are impaired in short tract gene conversion but maintain proficiency in sister chromatid exchange.     

BRCA2 and homologous recombination

Two recent papers provide new evidence relevant to the role of the breast cancer susceptibility gene BRCA2 in DNA repair. Moynahan et al provide genetic data indicating a requirement for BRCA2 in homology-dependent (recombinational) repair of DNA double-strand breaks. The second paper, by Davies et al, begins to address the mechanism through which BRCA2 makes its contribution to recombinational repair. BRCA2 appears to function in recombination via interactions with the major eukaryotic recombinase RAD51 [1,2,3]. We briefly review the context in which the two studies were carried out, we comment on the results presented, and we discuss models designed to account for the role of BRCA2 in RAD51-mediated repair.     

Mutation analysis of RAD51L1 (RAD51B/REC2) in multiple-case, non-BRCA1/2 breast cancer families

Although a significant proportion of familial aggregation of breast cancer remains unexplained, many of the currently known breast cancer susceptibility genes, including BRCA1, BRCA2 and TP53, play a role in maintaining genome integrity by engaging in DNA repair. RAD51L1 is one of the five RAD51 paralogs involved in homologous recombination (HR) repair of DNA double-strand breaks (DSBs); it also interacts directly with p53. Deleterious mutations have been found in one RAD51 paralog, RAD51C (RAD51L2), in non-BRCA1/2 breast and ovarian cancer families, which suggests that all five paralogs are strong candidate breast cancer susceptibility genes. A genome-wide association study (GWAS) has already identified a single nucleotide polymorphism (SNP) deep within intron 10 of RAD51L1 as a risk locus for breast cancer. Based on its biological functions and association with RAD51C, there is reason to suggest that RAD51L1 (RAD51B/REC2) may also contain high risk mutations in the gene that give rise to multiple-case breast cancer families. In order to investigate this hypothesis, we have used high resolution melt (HRM) analysis to screen RAD51L1 for germline mutations in 188 non-BRCA1/2 multiple-case breast cancer families and 190 controls. We identified a total of seven variants: one synonymous, three intronic, and three previously identified SNPs, but no truncating or nonsense changes. Therefore, our results suggest that RAD51L1 is unlikely to represent a high-penetrance breast cancer susceptibility gene.     

Secondary mutations of BRCA1/2 and drug resistance

Inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 cause increased risk of developing various cancers, especially breast and ovarian cancers. Tumors that develop in patients with inherited BRCA1/2 mutations are generally believed to be BRCA1/2-deficient. Cancer cells with BRCA1/2 deficiency are defective in DNA repair by homologous recombination and sensitive to interstrand DNA crosslinking agents, such as cisplatin and carboplatin, and poly(ADP-ribose) polymerase inhibitors. Therefore, these agents are logical choices for the treatment for BRCA1/2-deficient tumors and have shown to be clinically effective. However, BRCA1/2-mutated tumors often develop resistance to these drugs. Restoration of BRCA1/2 functions due to secondary BRCA1/2 mutations has been recognized as a mechanism of acquired resistance to cisplatin and poly(ADP-ribose) polymerase inhibitors in BRCA1/2-mutated cancer cells. This indicates that even disease-causing inherited mutations of tumor suppressor genes can be genetically reverted in cancer cells, if the genetic reversion is advantageous for the cells' survival. In this review, we will discuss this drug resistance mechanism.     

Suppression of homologous recombination sensitizes human tumor cells to IGF‐1R inhibition

Inhibition of type 1 IGF receptor (IGF‐1R) sensitizes to DNA‐damaging cancer treatments, and delays repair of DNA double strand breaks (DSBs) by non‐homologous end‐joining and homologous recombination (HR). In a recent screen for mediators of resistance to IGF‐1R inhibitor AZ12253801, we identified RAD51, required for the strand invasion step of HR. These findings prompted us to test the hypothesis that IGF‐1R‐inhibited cells accumulate DSBs formed at endogenous DNA lesions, and depend on residual HR for their repair. Indeed, initial experiments showed time‐dependent accumulation of γH2AX foci in IGF‐1R ‐inhibited or ‐depleted prostate cancer cells. We then tested effects of suppressing HR, and found that RAD51 depletion enhanced AZ12253801 sensitivity in PTEN wild‐type prostate cancer cells but not in cells lacking functional PTEN. Similar sensitization was induced in prostate cancer cells by depletion of BRCA2, required for RAD51 loading onto DNA, and in BRCA2^?/? colorectal cancer cells, compared with isogenic BRCA2^+/? cells. We also assessed chemical HR inhibitors, finding that RAD51 inhibitor BO2 blocked RAD51 focus formation and sensitized to AZ12253801. Finally, we tested CDK1 inhibitor RO‐3306, which impairs HR by inhibiting CDK1‐mediated BRCA1 phosphorylation. R0‐3306 suppressed RAD51 focus formation consistent with HR attenuation, and sensitized prostate cancer cells to IGF‐1R inhibition, with 2.4‐fold reduction in AZ12253801 GI₅₀ and 13‐fold reduction in GI₈₀. These data suggest that responses to IGF‐1R inhibition are enhanced by genetic and chemical approaches to suppress HR, defining a population of cancers (PTEN wild‐type, BRCA mutant) that may be intrinsically sensitive to IGF‐1R inhibitory drugs.     

Breast cancer risk reduction associated with the RAD51 polymorphism among carriers of the BRCA1 5382insC mutation in Poland.

The observed heterogeneity of breast cancer risk among women who carry the same BRCA1 mutation suggests the existence of modifying environmental and genetic factors. The product of the RAD51 gene functions with BRCA1 and BRCA2 in the repair of double-stranded DNA breaks. To establish whether polymorphic variation of RAD51 modifies risk for hereditary breast cancer, we conducted a matched case-control study on 83 pairs of female carriers of the BRCA1 5382insC mutation. Cases consisted of women with breast cancer, and controls were women with the same mutation but who were unaffected. The frequency of the RAD51 135C variant allele was established in cases and controls using RFLP-PCR. The RAD51 135C allele was detected in 37% of unaffected and in 17% of affected BRCA1 carriers. Among 27 discordant matched pairs, the RAD51 135C allele was found in the healthy carrier on 22 occasions and in the affected carrier on only five occasions (odds ratio = 0.23; 95% confidence interval, 0.07-0.62; P = 0.0015). This finding suggests that RAD51 is a genetic modifier of breast cancer risk in BRCA1 carriers in the Polish population. It will be of interest to confirm this in other populations as well.     

BRCA2 protects mammalian cells from heat shock

Purpose: Heat shock induces DNA double-strand breaks (DSBs) in mammalian cells. Mammalian cells are capable of repairing DSBs by utilising the homologous recombination (HR) pathway. Breast cancer susceptibility gene 2 (BRCA2) is known to regulate the HR pathway. Here, we investigate the role of BRCA2 in repairing DNA damage induced by heat shock.

Materials and methods: Chinese hamster lung fibroblast cell lines and human tongue squamous cell carcinoma SAS cells were used. RAD51 foci formation assay was used as an HR indicator. Heat sensitivity was analysed with colony forming assays. Phosphorylated histone H2AX (γH2AX) intensity, which correlates with the number of DSBs, was analysed with flow cytometry.

Results: RAD51 foci appeared with heat shock, and the number of cells with RAD51 foci was maximal at about 4?h after heat shock. Heat-induced RAD51 foci co-localised with γH2AX foci. BRCA2-deficient cells were sensitive to heat when compared to their parental wild-type cells. Heat-induced γH2AX was higher in BRCA2-deficient cells compared to parental cells. In SAS cells, cells transfected with BRCA2-siRNA were more sensitive to heat than cells transfected with negative control siRNA. Apoptotic bodies increased in number more rapidly in BRCA2-siRNA transfected cells than in cells transfected with negative control siRNA when cells were observed at 48?h after a heat treatment. In addition, cells deficient in BRCA2 were incapable of activating heat-induced G₂/M arrest.

Conclusion: BRCA2 has a protecting role against heat-induced cell death. BRCA2 might be a potential molecular target for hyperthermic cancer therapy.     

The roles of BRCA1 and BRCA2 and associated proteins in the maintenance of genomic stability

The BRCA1 and BRCA2 proteins are important in maintaining genomic stability by promoting efficient and precise repair of double-strand breaks. The main role of BRCA2 appears to involve regulating the function of RAD51 in the repair by homologous recombination. BRCA1 has a broader role upstream of BRCA2, participating in various cellular processes in response to DNA damage. The DNA repair defect associated with mutations in BRCA1 or BRCA2 could be exploited to develop new targeted therapeutic approaches for cancer occurring in mutation carriers.     

A single-nucleotide polymorphism in the RAD51 gene modifies breast cancer risk in BRCA2 carriers, but not in BRCA1 carriers or noncarriers

Variation in the penetrance estimates for BRCA1 and BRCA2 mutation carriers suggests that other genetic polymorphisms may modify the cancer risk in carriers. The RAD51 gene, which participates in homologous recombination double-strand breaks (DSB) repair in the same pathway as the BRCA1 and BRCA2 gene products, is a candidate for such an effect. A single-nucleotide polymorphism (SNP), RAD51-135g-->c, in the 5' untranslated region of the gene has been found to elevate breast cancer (BC) risk among BRCA2 carriers. We genotyped 309 BRCA1/2 mutation carriers, of which 280 were of Ashkenazi origin, 166 noncarrier BC patients and 152 women unaffected with BC (a control group), for the RAD51-135g-->c SNP. Risk analyses were conducted using COX proportional hazard models for the BRCA1/2 carriers and simple logistic regression analysis for the noncarrier case-control population. BRCA2 carriers were also studied using logistic regression and Kaplan-Meier survival analyses. The estimated BC hazard ratio (HR) for RAD51-135c carriers adjusted for origin (Ashkenazi vs non-Ashkenazi) was 1.28 (95% CI 0.85-1.90, P=0.23) for BRCA1/2 carriers, and 2.09 (95% CI 1.04-4.18, P=0.04) when the analysis was restricted to BRCA2 carriers. The median BC age was younger in BCRA2-RAD51-135c carriers (45 (95% CI 36-54) vs 52 years (95% CI 48-56), P=0.05). In a logistic regression analysis, the odds ratio (OR) was 5.49 (95% CI 0.5-58.8, P=0.163). In noncarrier BC cases, carrying RAD51-135c was not associated with BC risk (0.97; 95% CI 0.47-2.00). These results indicate significantly elevated risk for BC in carriers of BRCA2 mutations who also carry a RAD51-135c allele. In BRCA1 carriers and noncarriers, no effect for this SNP was found.