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.

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.

MSH2-deficient human cells exhibit a defect in the accurate termination of homology-directed repair of DNA double-strand breaks.

Mutations in the mismatch repair (MMR) genes hMSH2 and hMLH1 have been associated with hereditary nonpolyposis colorectal cancer. Tumor cell lines that are deficient in MMR exhibit a high mutation rate, a defect in the response to certain types of DNA damage and in transcription-coupled repair, as well as an increase in the rate of gene amplification. We show here that hMSH2-deficient tumor cell lines lost most of their ability to accurately repair plasmid DNA double-strand breaks (DSBs) by homologous recombination, compared with MMR-proficient or hMLH1-deficient tumor cell lines. In all of these cell lines, DSB repair occurred almost exclusively by nonreciprocal homologous recombination: gene conversion (GC). However, there were two types of GC products: precise and rearranged. The rearranged products contained deletions or insertions of sequences and represented GC intermediates trapped at various stages and shunted to nonhomologous end joining. In MMR-proficient or MLH1-deficient cells, >50% of GC products were of the precise type, whereas in two MSH2-deficient backgrounds, this proportion decreased to 8%, whereas that of rearranged GC products increased by 2-fold. These results seem to predict a novel way by which MSH2-deficiency could promote mutation: deletion or insertion mutations associated with DSB repair, which may also contribute to cancer predisposition.

Allelic transcripts dosage effect in morphologically normal ovarian cells from heterozygous carriers of a BRCA1/2 French Canadian founder mutation.

We hypothesized that the transcriptome of primary cultures of morphologically normal ovarian surface epithelial cells could be altered by the presence of a heterozygous BRCA1 or BRCA2 mutation. We aimed to discover early events associated with ovarian carcinogenesis, which could represent putative targets for preventive strategies of this silent killer tumor. We identified the first molecular signature associated with French Canadian BRCA1 or BRCA2 founder mutations in morphologically normal ovarian epithelial cells. We discovered that wild-type and mutated BRCA2 allelic transcripts were expressed not only in morphologically normal but also in tumor cells from BRCA2-8765delAG carriers. Further analysis of morphologically normal ovarian and tumor cells from BRCA1-4446C>T carriers lead to the same observation. Our data support the idea that one single hit in BRCA1 or BRCA2 is sufficient to alter the transcriptome of phenotypically normal ovarian epithelial cells. The highest level of BRCA2-mutated allele transcript expression was measured in cells originating from the most aggressive ovarian tumor. The penetrance of the mutation and the aggressiveness of the related tumor could depend on a dosage effect of the mutated allele transcript.

Disruption of p53 by the viral oncoprotein HPV16-E6 does not deregulate chromosomal homologous recombination in a transcriptional interference-free assay system.

In addition to its well established role in the maintenance of genome integrity by regulating transcription of genes involved in cell cycle arrest and programmed cell death, the tumour suppressor p53 has also been shown to inhibit spontaneous chromosomal homologous recombination (HR) between adjacent transcription units, raising the possibility that p53 may prevent chromosomal rearrangements by suppressing HR between repetitive DNA elements (ectopic HR). Consistent with its role in the maintenance of genome integrity is that p53 does not suppress HR between homologous chromosomes (allelic HR) or identical sister chromatids, raising the question of how p53 discriminates between ectopic and allelic HR events. Here, we report that disruption of human p53 by the viral oncoprotein HPV16-E6 does not result in increased rates of chromosomal HR between adjacent DNA repeats in a transcriptional interference-free assay system in which a HR reporter gene can escape transcription repression. These results argue against a direct role for p53 in the regulation of HR mechanisms, imply that HR assay systems may be important determinants of the outcome, and suggest that p53 may suppress ectopic HR through its known ability to repress transcription and alter chromatin structure.