Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52 ؊/؊ mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.

In meiotic prophase, synaptonemal complexes (SCs) closely appose homologous chromosomes (homologs) along their length. SCs are assembled from two axial elements (AEs), one along each homolog, which are connected by numerous transverse filaments (TFs In meiosis, two rounds of chromosome segregation follow one round of replication. The first segregation, meiosis I, is reductional, as homologous chromosomes (homologs) move to opposite poles, whereas meiosis II is equational, because sister chromatids disjoin. The disjunction of homologs is prepared during the prophase of meiosis I, when homologs pair and nonsister chromatids of homologs recombine (for review, see Zickler and Kleckner 1999). The resulting crossovers and cohesion between the sister chromatids connect the homologs and ensure their proper disjunction at meiosis I. In most analyzed eukaryotes, meiotic recombination is accompanied by the close apposition of homologs by a zipper-like proteinaceous structure, the synaptonemal complex (SC). After premeiotic S-phase, the two sister chromatids of each chromosome develop a common axial structure, the axial element (AE), which consists of a linear array of protein complexes involved in sister chromatid cohesion (cohesin complexes), associated with various additional proteins (for review, see Page and Hawley 2004). Numerous transverse filaments (TFs) then connect the AEs of two homologs (synapsis) to form an SC. Within the SC, AEs are called lateral elements (LEs). Genes encoding TF proteins have been identified in mammals (Sycp1), budding yeast (ZIP1), Drosophila (c(3)G), and Caenorhabditis (Syp-1 and Syp-2). SYCP1, Zip1, and C(3)G are long coiled-coil proteins with globular domains at both ends. Within SCs, they form parallel coiled-coil homodimers, which are embedded with their C termini in the LEs, whereas the N termini of TF protein molecules from opposite LEs overlap in the narrow region between the LEs of the two homologs. Caenorhabditis Syp-1 and Syp-2 are two short coiled-coil proteins, which possibly take the place of a single longer coiled-coil protein in other species (for review, see Page and Hawley 2004).In the three species in which it has been analyzed, Drosophila, Caenorhabditis, and yeast, TF-deficient mu-

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52 ؊/؊ ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.Double-strand breaks (DSBs) in the DNA of living organisms occur during several physiological processes including meiotic recombination, mating-type switching in yeast, and V(D)J rearrangement in developing B and T lymphocytes. Agents such as ionizing radiation and certain chemicals also lead to the induction of DSBs in the genome. If left unrepaired, DSBs result in broken chromosomes and cell death, as has been shown convincingly in yeast (5). Alternatively, incorrect repair of DSBs may generate deletions, chromosome rearrangements, and cell transformation and eventually lead to the formation of tumors.Two main pathways are known to be involved in the repair of DSBs in eukaryotes: end-to-end rejoining, a homology-independent but error-prone process, and error-free repair via (homologous) recombination. Repair of DSBs in the yeast Saccharomyces cerevisiae occurs predominantly via recombination, whereas a contribution of end-to-end rejoining can be observed only in a recombination-deficient background (9, 27, 47). Recombinational repair in S. cerevisiae involves the genes of the RAD52 epistasis group, of which nine members have been identified thus far (ScRAD50, ScRAD51, ScRAD52, ScRAD54, ScRAD55, ScRAD57, ScRAD59, ScMRE11, and ScXRS2) (2,11,15,16,44). Interestingly, it has been shown that ScRAD50, ScMRE11, and ScXRS2 are also involved in end-to-end rejoining (10,28,55). Mutations in genes of the RAD52 group result in an increased sensitivity to ionizing radiation and defects in one or more types of recombination. Among these mutants, the Scrad51, Scrad52, and Scrad54 mutants display the most severe radiation sensitivity and defects in recombination.Biochemical experiments with S. cerevisiae have shown that the ScRad51 protein forms nucleoprotein filaments with single-stranded DNA and promotes pairing and limited strand exchange (51). The ScRad52 protein alone or a heterodimer of ScRad55 and ScRad57 functions as a cofactor in this reaction, ...

NuRD is recruited to DNA double-strand breaks, where it promotes RNF8/RNF168 histone ubiquitylation and accumulation of DNA repair factors (see also related paper by Larsen et al. in this issue).