SUMMARY Inappropriate homologous recombination (HR) can cause gross chromosomal rearrangements that in mammalian cells may lead to tumorigenesis. In yeast, the Srs2 protein is an anti-recombinase that eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has proven to be elusive. In this work, we identify C. elegans SPAR-1 as a functional analogue of Srs2 and describe its vertebrate counterpart, SPAR1/RTEL1, which is required for genome stability and tumour avoidance. We find that spar-1 mutant worms and SPAR1 knockdown human cells share characteristic phenotypes with yeast srs2 mutants, including inviability upon deletion of the sgs1/BLM homologue, hyper-recombination, and DNA damage sensitivity. In vitro, purified human SPAR1 antagonises HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control following deregulation of SPAR1/RTEL1 may be a critical event that drives genome instability and cancer.

Meiotic crossovers (COs) are tightly regulated to ensure that COs on the same chromosome are distributed far apart (crossover interference, COI) and that at least one CO is formed per homolog pair (CO homeostasis). CO formation is controlled in part during meiotic double-strand break (DSB) creation in Caenorhabditis elegans, but a second level of control must also exist because meiotic DSBs outnumber COs. We show that the antirecombinase RTEL-1 is required to prevent excess meiotic COs, probably by promoting meiotic synthesis-dependent strand annealing. Two distinct classes of meiotic COs are increased in rtel-1 mutants, and COI and homeostasis are compromised. We propose that RTEL-1 implements the second level of CO control by promoting noncrossovers.

Homologous recombination (HR) is essential for the repair of blocked or collapsed replication forks and for the production of crossovers between homologs that promote accurate meiotic chromosome segregation. Here, we identify HIM-18, an ortholog of MUS312/Slx4, as a critical player required in vivo for processing late HR intermediates in Caenorhabditis elegans. DNA damage sensitivity and an accumulation of HR intermediates (RAD-51 foci) during premeiotic entry suggest that HIM-18 is required for HR–mediated repair at stalled replication forks. A reduction in crossover recombination frequencies—accompanied by an increase in HR intermediates during meiosis, germ cell apoptosis, unstable bivalent attachments, and subsequent chromosome nondisjunction—support a role for HIM-18 in converting HR intermediates into crossover products. Such a role is suggested by physical interaction of HIM-18 with the nucleases SLX-1 and XPF-1 and by the synthetic lethality of him-18 with him-6, the C. elegans BLM homolog. We propose that HIM-18 facilitates processing of HR intermediates resulting from replication fork collapse and programmed meiotic DSBs in the C. elegans germline.

Commentary 501 IntroductionMeiosis is the specialised reductive division that generates haploid cells. During this process, a single round of replication is followed by two rounds of chromosome segregation: in the first division (meiosis I), homologous chromosomes segregate, whereas in the second division, sister chromatids segregate (meiosis II). A key step in meiosis I is the recognition of homologous chromosomes, which then align and pair along the length of the chromosome. Once homologues have aligned, synapsis can proceed with the formation of the synaptonemal complex (SC), a protein structure that supports and maintains homologues in close juxtaposition and serves as a scaffold for crossover-promoting recombination factors. Meiotic crossing over involves the generation of meiotic doublestrand breaks (DSBs), which are subsequently repaired either as crossovers or non-crossovers (Fig. 1). Meiotic recombination is not only necessary to create new allele combinations that generate genetic diversity, but is also essential in ensuring accurate chromosome segregation at the first meiotic division because the crossover acts as a tether between homologues, which ensures that each homologue will properly align at the metaphase plate and thereby correctly attach to the spindle. DSB repair occurs concurrently with SC formation and is required for normal synapsis in yeast and mice (Baudat et al., 2000;Roeder, 1997;Romanienko and Camerini-Otero, 2000), whereas in Caenorhabditis elegans and Drosophila melanogaster, homologue pairing and SC formation can occur independently of meiotic recombination (Colaiacovo et al., 2003; Dernburg et al., 1998;Liu et al., 2002;McKim et al., 1998).The process of meiotic recombination is initiated when meiotic DSBs are created by the endonuclease SPO11, in conjunction with a number of additional proteins (Keeney and Neale, 2006). DSBs are then resected to generate 3Ј single-strand DNA (ssDNA) overhangs that are initially bound by replication protein A (RPA), which is subsequently displaced by the recombinase radiation sensitive 51 (RAD51) and/or the meiosis-specific recombinase dosage suppressor of Mck1 (DMC1) to form nucleoprotein filaments. These filaments serve to find a complimentary sequence within a homologous chromosome, at which they instigate singleend strand invasions (Hunter and Kleckner, 2001) to generate socalled displacement loop (D loop) recombination intermediates ( Fig. 1). If the second end of the original DSB binds with the homologous chromosome, a double Holliday junction is formed, which can be resolved to generate either a non-crossover or an interhomologue crossover, the latter of which is hereafter referred to simply as crossover (Bishop and Zickler, 2004;Schwacha and Kleckner, 1995). Double Holliday junctions can also be processed through dissolution, which results in a non-crossover ( Fig. 1) (Wu and Hickson, 2003). Meiotic non-crossovers have also been proposed to form when strand invasion is transient, and when a limited amount of DNA synthesis occurs befor...

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by defective DNA interstrand cross-link (ICL) repair. Here, we show that DOG-1 is the Caenorhabditis elegans homologue of FANCJ, a helicase mutated in FA-J patients. DOG-1 performs a conserved role in ICL repair, as dog-1 mutants are hypersensitive to ICL-inducing agents, but not to UVC irradiation or X rays. Genetic analysis indicated that dog-1 is epistatic with fcd-2 (C. elegans FANCD2) but is nonepistatic with brc-1 (C. elegans BRCA1), thus establishing the existence of two distinct pathways of ICL repair in worms. Furthermore, DOG-1 is dispensable for FCD-2 and RAD-51 focus formation, suggesting that DOG-1 operates downstream of FCD-2 and RAD-51 in ICL repair. DOG-1 was previously implicated in poly(G)/poly(C) (G/C) tract maintenance during DNA replication. G/C tracts remain stable in the absence of ATL-1, CLK-2 (FA pathway activators), FCD-2, BRC-2, and MLH-1 (associated FA components), implying that DOG-1 is the sole FA component required for G/C tract maintenance in a wild-type background. However, FCD-2 is required to promote deletion-free repair at G/C tracts in dog-1 mutants, consistent with a role for FA factors at the replication fork. The functional conservation between DOG-1 and FANCJ suggests a possible role for FANCJ in G/C tract maintenance in human cells.Fanconi anemia (FA) is a rare chromosomal instability syndrome associated with various congenital abnormalities, bone marrow failure, and susceptibility to cancer (24). FA cells exhibit a characteristic cellular hypersensitivity to agents that cause DNA interstrand cross-links (ICLs), such as UV-activated trimethylpsoralen (TMP), nitrogen mustard, cisplatin, mitomycin C, and diepoxybutane, indicating that the FA proteins function in repair of ICLs (reviewed in references 24 and 31). Although ICLs are one of the most cytotoxic DNA lesions, the function of the FA factors in ICL repair is not well defined.Currently, 13 FA complementation groups have been identified (A, B, C, D1/BRCA2, D2, E, F, G, I, J/BRIP1, L, M, and N) (34, 39), and more FA-associated genes are likely to be discovered, as a number of patients cannot be assigned to these groups. Eight of these proteins (FANCA, -B, -C, -E, -F, -G, -L, and -M) function in the FA core complex, along with additional proteins, such as FAAP100, for which no corresponding patients have been identified (27). The core complex is required for monoubiquitylation of FANCD2 and FANCI (38,39). Monoubiquitylation appears to trigger the recruitment of FANCD2 and FANCI to DNA damage foci (24,37,38), where FANCD2 is known to interact with other repair proteins, including BRCA1, BRCA2, and the MRE11/RAD50/NBS1 complex (21). BRCA2 and FANCJ are thought to act downstream of FANCD2 monoubiquitylation in the FA pathway, as FANCD2 modification and recruitment are not affected in BRCA2-or FANCJ-deficient cells (24). While BRCA2/ FANCD1 appear to regulate the assembly and disassembly of RAD51 onto single-stranded DNA during recombinational repair (17,...

In C. elegans, DOG-1 prevents deletions that initiate in polyG/polyC tracts (G/C tracts), most likely by unwinding secondary structures that can form in G/C tracts during lagging-strand DNA synthesis. We have used the dog-1 mutant to assay the in vivo contribution of various repair genes to the maintenance of G/C tracts. Here we show that DOG-1 and the BLM ortholog, HIM-6, act synergistically during replication; simultaneous loss of function of both genes results in replicative stress and an increase in the formation of small deletions that initiate in G/C tracts. Similarly, we demonstrate that the C. elegans orthologs of the homologous recombination repair genes BARD1, RAD51, and XPF and the trans-lesion synthesis polymerases polh and polk contribute to the prevention of deletions in dog-1 mutants. Finally, we provide evidence that the small deletions generated in the dog-1 background are not formed through homologous recombination, nucleotide excision repair, or nonhomologous end-joining mechanisms, but appear to result from a mutagenic repair mechanism acting at G/C tracts. Our data support the hypothesis that absence of DOG-1 leads to replication fork stalling that can be repaired by deletion-free or deletion-prone mechanisms.

Telomere maintenance and DNA repair are crucial processes that protect the genome against instability. RTEL1, an essential iron–sulfur cluster-containing helicase, is a dominant factor that controls telomere length in mice and is required for telomere integrity. In addition, RTEL1 promotes synthesis-dependent strand annealing to direct DNA double-strand breaks into non-crossover outcomes during mitotic repair and in meiosis. Here, we review the role of RTEL1 in telomere maintenance and homologous recombination and discuss models linking RTEL1’s enzymatic activity to its function in telomere maintenance and DNA repair.

The generation and resolution of joint molecule recombination intermediates is required to ensure bipolar chromosome segregation during meiosis. During wild type meiosis in Caenorhabditis elegans, SPO-11-generated double stranded breaks are resolved to generate a single crossover per bivalent and the remaining recombination intermediates are resolved as noncrossovers. We discovered that early recombination intermediates are limited by the C. elegans BLM ortholog, HIM-6, and in the absence of HIM-6 by the structure specific endonuclease MUS-81. In the absence of both MUS-81 and HIM-6, recombination intermediates persist, leading to chromosome breakage at diakinesis and inviable embryos. MUS-81 has an additional role in resolving late recombination intermediates in C. elegans. mus-81 mutants exhibited reduced crossover recombination frequencies suggesting that MUS-81 is required to generate a subset of meiotic crossovers. Similarly, the Mus81-related endonuclease XPF-1 is also required for a subset of meiotic crossovers. Although C. elegans gen-1 mutants have no detectable meiotic defect either alone or in combination with him-6, mus-81 or xpf-1 mutations, mus-81;xpf-1 double mutants are synthetic lethal. While mus-81;xpf-1 double mutants are proficient for the processing of early recombination intermediates, they exhibit defects in the post-pachytene chromosome reorganization and the asymmetric disassembly of the synaptonemal complex, presumably triggered by crossovers or crossover precursors. Consistent with a defect in resolving late recombination intermediates, mus-81; xpf-1 diakinetic bivalents are aberrant with fine DNA bridges visible between two distinct DAPI staining bodies. We were able to suppress the aberrant bivalent phenotype by microinjection of activated human GEN1 protein, which can cleave Holliday junctions, suggesting that the DNA bridges in mus-81; xpf-1 diakinetic oocytes are unresolved Holliday junctions. We propose that the MUS-81 and XPF-1 endonucleases act redundantly to process late recombination intermediates to form crossovers during C. elegans meiosis.