Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining.

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.

Repair bias of large loop mismatches during recombination in mammalian cells depends on loop length and structure.

Repair of loop mismatches was investigated in wild-type and mismatch binding-defective Chinese hamster ovary (CHO) cells. Loop mismatches were formed in vivo during extrachromosomal recombination between heteroallelic plasmid substrates. Recombination was expected to occur primarily by single-strand annealing (SSA), yielding 12- or 26-base nonpalindromic loop mismatches, and 12-, 26-, or 40-base palindromic loop mismatches. Nonpalindromic loops were repaired efficiently and with bias toward loop loss. In contrast, the 12-base palindromic loop was repaired with bias toward loop retention, indicating that repair bias depends on loop structure. Among the palindromic loops, repair bias was dependent on loop length, with bias shifting from loop retention to loop loss with increasing loop size. For both palindromic and nonpalindromic loops, repair efficiencies and biases were independent of the general (MSH/MLH) mismatch repair pathway. These results are discussed with respect to the maintenance of large nonpalindromic insertions, and of small and large palindromes, in eukaryotic genomes.

Dissociation of p53-mediated suppression of homologous recombination from G1/S cell cycle checkpoint control.

The tumor suppressor p53 is considered as the guardian of the genome which is activated following genotoxic stress. In many cell types, p53 mediates G1 cell cycle arrest as the predominant cellular response. Inactivation of wild-type p53 leads to loss of G1/S checkpoint control and to genomic instability, including increased spontaneous homologous recombination (HR). To determine whether regulation of the G1/S checkpoint is required for suppression of HR, we assessed recombination events using a plasmid substrate that stably integrated into the genome of p53-null mouse fibroblasts. Exogenous expression of a temperature-sensitive p53 protein (Ala135 to Val), which had lost trans-activation function and could not regulate G1/S transition when in mutant conformation, reduced HR rates to the same extent as wild-type p53. Furthermore, a p53 construct with an alternatively-spliced carboxy terminus also retained this ability in the absence of both activities, G1/S control and non-sequence specific DNA binding as mediated by the carboxy terminus. Our data dissociate regulation of HR by p53 from its role as a cell cycle checkpoint protein. The results support a model which extends p53's role as a guardian of the genome to include transactivation-independent regulatory functions in DNA repair, replication and recombination.

Biased short tract repair of palindromic loop mismatches in mammalian cells.

Mismatch repair of palindromic loops in the presence or absence of single-base mismatches was investigated in wild-type and mismatch-binding defective mutant Chinese hamster ovary cells. Recombination intermediates with a maximum heteroduplex DNA (hDNA) region of 697 bp contained a centrally located, phenotypically silent 12-base palindromic loop mismatch, and/or five single-base mismatches. In wild-type cells, both loops and single-base mismatches were efficiently repaired (80-100%). When no other mismatches were present in hDNA, loops were retained with a 1.6-1.9:1 bias. However, this bias was eliminated when single-base mismatches were present, perhaps because single-base mismatches signal nick-directed repair. In the multiple marker crosses, most repair tracts were long and continuous, with preferential loss of markers in cis to proximal nicks, consistent with nicks directing most repair in this situation. However, approximately 25% of repair tracts were discontinuous as a result of loop-specific repair, or from segregation or short tract repair of single-base mismatches. In mutant cells, single-base mismatches were repaired less frequently, but the loop was still repaired efficiently and with bias toward loop retention, indicating that the defect in these cells does not affect loop-specific repair. Repair tracts in products from mutant cells showed a wide variety of mosaic patterns reflecting short regions of repair and segregation consistent with reduced nick-directed repair. In mutant cells, single-base mismatches were repaired more efficiently in the presence of the loop than in its absence, a likely consequence of corepair initiated at the loop.

Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.

Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.