Homologous recombination (HR) is a central process to ensure genomic stability in somatic cells and during meiosis. HR-associated DNA synthesis determines in large part the fidelity of the process. A number of recent studies have demonstrated that DNA synthesis during HR is conservative, less processive, and more mutagenic than replicative DNA synthesis. In this review, we describe mechanistic features of DNA synthesis during different types of HR-mediated DNA repair, including synthesis-dependent strand annealing, break-induced replication, and meiotic recombination. We highlight recent findings from diverse eukaryotic organisms, including humans, that suggest both replicative and translesion DNA polymerases are involved in HR-associated DNA synthesis. Our focus is to integrate the emerging literature about DNA polymerase involvement during HR with the unique aspects of these repair mechanisms, including mutagenesis and template switching.

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.DNA repair | genome stability | translocation D NA double-strand breaks (DSBs) are toxic lesions that can be repaired by two major pathways in eukaryotes: nonhomologous end-joining (NHEJ) and homologous recombination (HR) (1). Although HR repairs DSBs in a template-dependent, high-fidelity manner, NHEJ functions to ligate DSB ends together using no or very short (1-4 bp) homology. Recently, a new pathway was identified in eukaryotes, which uses microhomologies (MHs) to repair a DSB and does not require the central proteins used in HR (Rad51, Rad52) or NHEJ (Ku70-Ku80) (2-5). In mammalian cells, this pathway of repair is known as alternative end-joining (Alt-EJ) and is often but not always associated with MHs, whereas in budding yeast, the commensurate pathway, MH-mediated end-joining (MMEJ), will typically use 5-25 bp of MH (6, 7). These pathways are associated with genomic rearrangements, and cancer genomes show evidence of MH-mediated rearrangements (8-12). In addition, eukaryotic genomes contain many dispersed repetitive elements that can lead to genome rearrangements when recombination occurs between them (13-16). Therefore, controlling DSB repair in the human genome, which features a variety of repeats, is especially important given the fact that recombination between repetitive elements has been implicated in genomic instability associated with disease (17-20).The original characterization of Alt-EJ in mammalian cells suggested it did not rep...

Using a model system, we have shown that replicative senescence is accompanied by a 16-fold increase in base substitution and frameshift mutations near a chromosome end. The increase was dependent on errorprone polymerases required for the mutagenic response to DNA lesions that block the replication fork.S ACCHAROMYCES cerevisiae cells lacking telomerase, a ribonucleoprotein complex required for telomere replication, experience progressive telomere degradation that culminates in replicative senescence (Lendvay et al. 1996). Deletions that encompass the CAN1 locus located $32 kb from the telomere on the left arm of chromosome V accumulated during senescence (Hackett et al. 2001;Hackett and Greider 2003) and have been attributed to replication fork stalling (Motegi et al. 2006). Since replication fork stalling also generates mutations (Quah et al. 1980), we investigated the effect of telomere dysfunction on the generation of mutations at the CAN1 locus.Mutation rate analysis: We examined the behavior of mutants defective for EST2, which encodes the catalytic subunit of telomerase (Counter et al. 1997) (Table 1). We determined the mutation rate at the CAN1 locus using an assay that selects against deletions in serial cultures of wild-type and est2 mutant cells. We observed no significant change from the wild-type rate of CAN1 mutation in est2 cultures before senescence (P ¼ 0.07), a 16-fold increase during senescence (P , 0.0001), and the restoration of wild-type levels upon recovery (P ¼ 0.09) ( Figure 1A). No senescence-dependent changes in the mutation rate at the CYH2 locus, located 310 kb from its telomere on the left arm of chromosome VII, were observed in est2 mutant cultures before (P ¼ 0.68), during (P ¼ 1.0), or after replicative senescence (P ¼ 1.0) (Figure 2), suggesting that the mutagenic effect is restricted to telomere-proximal sequences. The can1 mutation spectrum observed for senescent est2 mutant cells was similar to that of wild type ( Table 2), suggesting that the mechanism of senescence-dependent mutagenesis in est2 cells may be similar to the mechanism of spontaneous mutagenesis in wild-type cells. Since 50-70% of spontaneous mutagenesis has been attributed to the action of error-prone polymerases (Quah et al. 1980), we investigated whether they were involved in the mechanism underlying senescence-dependent mutagenesis. We examined the effects of mutations in the RAD30, REV7, and REV1 genes, which are required for error-prone polymerase function in yeast (Haracska et al. 2000;Johnson et al. 2000;Goodman 2002;Prakash et al. 2005). Mutation rates in est2 rad30 cultures before (P ¼ 0.32), during (P ¼ 0.34), and after senescence (P ¼ 0.15) were not significantly different from those in est2 cultures (Figure 1, A and B), suggesting that Pol h does not contribute to senescencedependent mutagenesis. In contrast, the rev1 and rev7 mutations completely suppressed senescence-dependent mutagenesis, as the CAN1 mutation rates were not significantly different from those of rev1 and rev7 mutants bef...

Telomerase is a ribonucleoprotein complex required for the replication and protection of telomeric DNA in eukaryotes. Cells lacking telomerase undergo a progressive loss of telomeric DNA that results in loss of viability and a concomitant increase in genome instability. We have used budding yeast to investigate the relationship between telomerase deficiency and the generation of chromosomal translocations, a common characteristic of cancer cells. Telomerase deficiency increased the rate of formation of spontaneous translocations by homologous recombination involving telomere proximal sequences during crisis. However, telomerase deficiency also decreased the frequency of translocation formation following multiple HO-endonuclease catalyzed DNA double-strand breaks at telomere proximal or distal sequences before, during and after crisis. This decrease correlated with a sequestration of the central homologous recombination factor, Rad52, to telomeres determined by chromatin immuno-precipitation. This suggests that telomerase deficiency results in the sequestration of Rad52 to telomeres, limiting the capacity of the cell to repair double-strand breaks throughout the genome. Increased spontaneous translocation formation in telomerase-deficient yeast cells undergoing crisis is consistent with the increased incidence of cancer in elderly humans, as the majority of our cells lack telomerase. Decreased translocation formation by recombinational repair of double-strand breaks in telomerase-deficient yeast suggests that the reemergence of telomerase expression observed in many human tumors may further stimulate genome rearrangement. Thus, telomerase may exert a substantial effect on global genome stability, which may bear significantly on the appearance and progression of cancer in humans.