SummaryEukaryotic genomes harbor a large number of homologous repeat sequences that are capable of recombining at high frequency. Their potential to disrupt genome stability highlights the need to understand how homologous recombination processes are coordinated. The S. cerevisiae Rad1-Rad10 endonuclease performs an essential role in recombination between repeated sequences by processing 3′ single-stranded intermediates formed during single-strand annealing and gene conversion events. Several recent studies have focused on factors involved in Rad1-Rad10-dependent removal of 3′ nonhomologous tails during homologous recombination, including Msh2-Msh3, Slx4, and the newly identified Saw1 protein (1-4). In addition, these studies suggest mechanisms for how DNA repair is coordinated by the DNA damage checkpoint machinery (1). This review aims to integrate these new findings with previous work to create a comprehensive model for how DNA repair and checkpoint factors act in concert to process 3′ nonhomologous tail intermediates that arise during homologous recombination. KeywordsRad1-Rad10; Saw1; Slx4; Msh2-Msh3; nonhomologous tail Homologous stretches of DNA sequences scattered throughout the genome are a threat to genome stability because of their potential to recombine. It is estimated that nearly half of the human genome consists of repetitive DNA (5,6), and genome rearrangements caused by recombination between repeats are known to contribute to a variety of human diseases, including many cancers (7)(8)(9). Repetitive sequences such as Alu elements are particularly susceptible to non-conservative homologous recombination via single-strand annealing (SSA), which results in the deletion of sequences located between the repeats (10-12). The abundance of such sequences in the human genome underscores the need for a comprehensive understanding of the homologous recombination processes that act on them.SSA is a major recombination pathway for repairing spontaneous and induced double-strand breaks (DSBs) that arise between repeated sequences (10,13,14). During SSA in S. cerevisiae, DSBs are resected 5′ to 3′ to reveal single-stranded homologous sequences ( Figure 1). Following Rad52-and Rad59-dependent annealing of the homologous sequences, the 3′ single-stranded DNA ends are nonhomologous to the new flanking regions, and must be cleaved in order to complete repair of the broken strands. The Rad1-Rad10 endonuclease and Msh2-Msh3 mismatch recognition complex are required for cleaving 3′ single-stranded nonhomologous tails on either side of the annealed region (15)(16)(17)(18)(19) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript been removed, SSA is completed by DNA synthesis initiated off of the newly cleaved 3′ ends followed by ligation (reviewed in 10).Rad1-Rad10 is a structure-specific endonuclease that functions in both nucleotide excision repair (NER) and homologous recombination (10,11,19,20). The significance of the role of Rad1-Rad10 in recombination is demonstrated by the severe developm...

The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

Efficient repair of DNA double-strand breaks (DSBs) requires the coordination of checkpoint signaling and enzymatic repair functions. To study these processes during gene conversion at a single chromosomal break, we monitored mating-type switching in Saccharomyces cerevisiae strains defective in the Rad1-Rad10-Slx4 complex. Rad1-Rad10 is a structure-specific endonuclease that removes 39 nonhomologous singlestranded ends that are generated during many recombination events. Slx4 is a known target of the DNA damage response that forms a complex with Rad1-Rad10 and is critical for 39-end processing during repair of DSBs by single-strand annealing. We found that mutants lacking an intact Rad1-Rad10-Slx4 complex displayed RAD9-and MAD2-dependent cell cycle delays and decreased viability during mating-type switching. In particular, these mutants exhibited a unique pattern of dead and switched daughter cells arising from the same DSB-containing cell. Furthermore, we observed that mutations in post-replicative lesion bypass factors (mms2D, mph1D) resulted in decreased viability during mating-type switching and conferred shorter cell cycle delays in rad1D mutants. We conclude that Rad1-Rad10-Slx4 promotes efficient repair during gene conversion events involving a single 39 nonhomologous tail and propose that the rad1D and slx4D mutant phenotypes result from inefficient repair of a lesion at the MAT locus that is bypassed by replication-mediated repair.

Summary Testicular germ cell tumors (TGCTs) are among the most responsive solid cancers to conventional chemotherapy. To elucidate the underlying mechanisms, we developed a mouse TGCT model featuring germ cell-specific Kras activation and Pten inactivation. The resulting mice developed malignant, metastatic TGCTs composed of teratoma and embryonal carcinoma, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4. Consistent with epidemiological data linking human testicular cancer risk to in utero exposures, embryonic germ cells were susceptible to malignant transformation, whereas adult germ cells underwent apoptosis in response to the same oncogenic events. Treatment of tumor-bearing mice with genotoxic chemotherapy not only prolonged survival and reduced tumor size, but selectively eliminated the OCT4-positive cancer stem cells. We conclude that the chemosensitivity of TGCTs derives from the sensitivity of their cancer stem cells to DNA-damaging chemotherapy.

Single-strand annealing (SSA) is an important homologous recombination mechanism that repairs DNA double strand breaks (DSBs) occurring between closely spaced repeat sequences. During SSA, the DSB is acted upon by exonucleases to reveal complementary sequences that anneal and are then repaired through tail clipping, DNA synthesis, and ligation steps. In baker's yeast, the Msh DNA mismatch recognition complex and the Sgs1 helicase act to suppress SSA between divergent sequences by binding to mismatches present in heteroduplex DNA intermediates and triggering a DNA unwinding mechanism known as heteroduplex rejection. Using baker's yeast as a model, we have identified new factors and regulatory steps in heteroduplex rejection during SSA. First we showed that Top3-Rmi1, a topoisomerase complex that interacts with Sgs1, is required for heteroduplex rejection. Second, we found that the replication processivity clamp proliferating cell nuclear antigen (PCNA) is dispensable for heteroduplex rejection, but is important for repairing mismatches formed during SSA. Third, we showed that modest overexpression of Msh6 results in a significant increase in heteroduplex rejection; this increase is due to a compromise in Msh2-Msh3 function required for the clipping of 39 tails. Thus 39 tail clipping during SSA is a critical regulatory step in the repair vs. rejection decision; rejection is favored before the 39 tails are clipped. Unexpectedly, Msh6 overexpression, through interactions with PCNA, disrupted heteroduplex rejection between divergent sequences in another recombination substrate. These observations illustrate the delicate balance that exists between repair and replication factors to optimize genome stability.