Caenorhabditis elegans HIM-18/SLX-4 Interacts with SLX-1 and XPF-1 and Maintains Genomic Integrity in the Germline by Processing Recombination Intermediates

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

Roles of the DOG-1 and JRH-1 helicase-like proteins in DNA repair in Caenorhabditis elegans

Helicases perform vital roles in the cell by unwinding D N A to make it accessible for the essential processes of replication, transcription and repair. In Caenorhabditis elegans, the DOG- 1 helicase-like protein is required for polyG/polyC-tract (G/C-tract) maintenance, as dog-l animals have a mutator phenotype characterized by deletions that initiate in G/C-tracts. DOG-1 may unwind secondary structures that form in polyguanine D N A during lagging strand replication. In order to more completely understand the role of dog-1, genetic interactors were identified, dog-1 functionally interacts with the him-6/BLM helicase. Absence of recombinational repair-implicated proteins in the dog-1 background, including HIM-6/BLM, RAD-51, BRD-1/BARD1 and HIM-9/XPF, as well as the trans-lesion synthesis polymerases polKMD po/7 increased the frequency of animals with G/C-tract deletions, indicating that these pathways are important mechanisms for repair at G/C-tracts in the absence of DOG-1. These data support the hypothesis that persisting D N A secondary structures can cause replication fork stalling, which can be resolved by deletion-free or deletion-prone mechanisms. DOG-1 has highest sequence identity to human BR1P1/FANCJ, which is mutated in patients from the Fanconi Anemia (FA) subgroup J. D N A damage sensitivity experiments indicated that, like chicken F A N C J cells, dog-1 mutants were not significantly sensitive to DNA damage from X-ray or UV-irradiation, but were extremely hypersensitive to the D N A interstrand cross-linking agent UVA-activated trimethylpsoralen. Thus, DOG-1 appears to have a conserved role in cross-link repair and is the C. elegans F A N C J homolog. Characterization of the dog-1/FANCJ-relatsd helicase, Jrh-1, revealed that mutants for this putative helicase are moderately sensitive to cross-linking agents, dog-1 jrh-1 double mutants displayed a synthetic lethal phenotype characterized by excessive recombination intermediates and mitotic catastrophe in the germline. However, absence of JRH-1 did not have any effect on G/C-tract deletions, indicating that JRH-1 does not have a redundant function with DOG-1 at G/C-tracts. Absence of JRH-1 reduced the fitness of eTl and nTl translocation hétérozygotes, but not translocation homozygotes, jrh-1 was synthetically lethal with him-6/BLM and with the endonuclease mus-81, suggesting a possible role for JRH-1 in regulating the balance between different types of repair.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Homologous Recombination Is Required for Genome Stability in the Absence of DOG-1 in Caenorhabditis elegans

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 polη and polκ 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

DOG-1 Is the Caenorhabditis elegans BRIP1/FANCJ Homologue and Functions in Interstrand Cross-Link Repair▿

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

Joint molecule resolution requires the redundant activities of MUS-81 and XPF-1 during Caenorhabditis elegans meiosis.

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

Joint Molecule Resolution Requires the Redundant Activities of MUS-81 and XPF-1 during <i>Caenorhabditis elegans</i> Meiosis

<div><p>The generation and resolution of joint molecule recombination intermediates is required to ensure bipolar chromosome segregation during meiosis. During wild type meiosis in <i>Caenorhabditis elegans</i>, 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 <i>C. elegans</i> 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 <i>C. elegans</i>. <i>mus-81</i> 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 <i>C. elegans gen-1</i> mutants have no detectable meiotic defect either alone or in combination with <i>him-6</i>, <i>mus-81</i> or <i>xpf-1</i> mutations, <i>mus-81;xpf-1</i> double mutants are synthetic lethal. While <i>mus-81;xpf-1</i> 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, <i>mus-81; xpf-1</i> 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 <i>mus-81; xpf-1</i> 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 <i>C. elegans</i> meiosis.</p></div

MUS-81 and HIM-6 prevent the accumulation of recombination intermediates and chromosome instability.

<p>(A) Distribution of RAD-51 foci in WT, <i>mus-81</i>, <i>him-6</i>, and <i>mus-81; him-6</i> animals. Zone definitions: 1 Early mitotic 2 Late mitotic 3 Transition 4 Early pachytene 5 Mid pachytene 6 Late pachytene 7 Diplotene. (B) Mean number of RAD-51 foci per nucleus from A. (C) Average number of DAPI-stained bodies in diakinesis oocytes of WT, <i>mus-81</i>, <i>him-6</i>, and <i>mus-81; him-6</i> animals. (D) Representative images of DAPI-stained bodies in diakinesis oocytes of WT, <i>mus-81</i>, <i>him-6</i>, and <i>mus-81; him-6</i> animals. Bars 2 µm.</p

MUS-81 and XPF-1 do not act redundantly to limit the formation of recombination intermediates.

<p>(A) Distribution of RAD-51 foci in WT, <i>mus-81</i>, <i>xpf-1</i>, and <i>mus-81; xpf-1</i> animals. Data for WT, <i>mus-81</i>, and <i>him-6</i> repeated from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003582#pgen-1003582-g001" target="_blank">Figure 1</a> for ease of comparison. Zone definitions: 1 Early mitotic 2 Late mitotic 3 Transition 4 Early pachytene 5 Mid pachytene 6 Late pachytene 7 Diplotene. (B) Average number of RAD-51 foci per nucleus from A. (C) Average number of DAPI-stained bodies in diakinesis oocytes of WT, <i>mus-81</i>, <i>xpf-1</i>, and <i>mus-81; xpf-1</i> animals. (D) Representative images of DAPI-stained bodies in diakinesis oocytes of WT, <i>mus-81; xpf-1</i>, and <i>mus-81; xpf-1</i> animals. Bars 2 µm.</p