SLX-1 Is Required for Maintaining Genomic Integrity and Promoting Meiotic Noncrossovers in the Caenorhabditis elegans Germline

Although the SLX4 complex, which includes structure-specific nucleases such as XPF, MUS81, and SLX1, plays important roles in the repair of several kinds of DNA damage, the function of SLX1 in the germline remains unknown. Here we characterized the endonuclease activities of the Caenorhabditis elegans SLX-1-HIM-18/SLX-4 complex co-purified from human 293T cells and determined SLX-1 germline function via analysis of slx-1(tm2644) mutants. SLX-1 shows a HIM-18/SLX-4–dependent endonuclease activity toward replication forks, 5′-flaps, and Holliday junctions. slx-1 mutants exhibit hypersensitivity to UV, nitrogen mustard, and camptothecin, but not gamma irradiation. Consistent with a role in DNA repair, recombination intermediates accumulate in both mitotic and meiotic germ cells in slx-1 mutants. Importantly, meiotic crossover distribution, but not crossover frequency, is altered on chromosomes in slx-1 mutants compared to wild type. This alteration is not due to changes in either the levels or distribution of double-strand breaks (DSBs) along chromosomes. We propose that SLX-1 is required for repair at stalled or collapsed replication forks, interstrand crosslink repair, and nucleotide excision repair during mitosis. Moreover, we hypothesize that SLX-1 regulates the crossover landscape during meiosis by acting as a noncrossover-promoting factor in a subset of DSBs

The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis

Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation.The Lalor Foundation Saravanapriah Nadarajan, Human Frontier Science Program Yonatan B Tzur, National Institutes of Health R01GM086434 Michael D Blower MRC-Core funding Enrique Martinez-Perez National Institutes of Health AG011085 J Wade Harper National Institutes of Health R01GM072551 Monica P ColaiacovoPeer reviewe

Plate phenotypes.

<p>Parentheses indicate the total number of:</p>a<p>singled hermaphrodites for which entire brood sizes were scored,</p>b<p>fertilized eggs scored,</p>c<p>adults scored,</p>d<p>L1-L4 worms,</p>e<p>data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002888#pgen.1002888-Saito1" target="_blank">[16]</a>.</p><p>ND, not determined due to n = 1.</p

<i>slx-1</i>(<i>tm2644</i>) mutants express splice variants that lack the C-terminus region required for an interaction with HIM-18, therefore encoding for nuclease-negative variants of SLX-1.

<p>(A) Schematic representation of the <i>slx-1</i> gene structure. The locations of the nuclease (red) and the PHD finger (blue) domains are shown. R202 and E243 are known catalytic sites of the GIY-YIG nuclease domain. The region deleted in <i>tm2644</i> is indicated. (B) <i>slx-1</i> cDNA in wild type. (C) <i>slx-1</i>(<i>tm2644</i>) variant cDNAs. Only start to stop codons are depicted. The number of times each splice variant was detected is indicated in parentheses at the right. (D) The chart indicates the frequency of cDNA variants observed in <i>slx-1(tm2644)</i> mutants. Blue, cDNAs including an intact PHD finger domain (exons 6 and 7). Pink, cDNAs including an intact nuclease domain (exons 3 and 4). Gray, cDNAs carrying neither PHD finger nor nuclease domains. (E) Yeast two-hybrid analysis of interactions between full-length HIM-18 fused to GAL4-AD and full-length or truncated SLX-1 fused to GAL4-DB. Interactions were scored by growth on –HIS+1 mM 3-AT plates.</p

<i>C. elegans</i> SLX-1 cleaves branched substrates in a HIM-18–dependent manner.

<p>(A) Protein constructs used in this study. Both the length of amino acid residues and domains are shown. FL: full length; CCD: conserved C-terminal domain; SBD: SLX1 binding domain; CD: catalytic dead; ZF: zinc finger; BTB: Broad-Complex, Tramtrack and Bric a brac; SAP: SAF-A/B, Acinus and PIAS; HtH: helix turn helix; NUC: nuclease; PHD: plant homeo domain. (B, C) HIM18 associates with full-length SLX1. (B) The indicated HA- and myc-tagged constructs were transfected into HEK293T cells, the cells lysed and the lysates immuno-precipitated with α-HA resin (Sigma) and immuno-blotted with the indicated antibodies. (C) As in (B), but the lysates were immunoprecipitated with α-myc resin (Sigma). (D–F) Cleavage activity of HIM-18/SLX-1. (D) The indicated proteins were transiently expressed and immuno-purified from 293T cells, incubated with a 32P-end-labeled replication fork (RF) substrate, the products were separated by native gel electrophoresis and visualized by autoradiography. The * indicates the labeled strand. The RF substrate and its major cleavage product are indicated to the left of the gel. (E) Similar to (D), but the Holliday Junction (HJ) was used as the substrate. The HJ substrate and its major cleavage products are indicated to the left of the gel. (F) Time-course to evaluate the cleavage rate of branched DNA substrates by HIM-18/SLX-1. Left panel: The HA-HIM-18CCD/myc-SLX-1 complex was transiently expressed and immuno-purified from 293T cells, incubated with the indicated substrates, the reaction was stopped at the indicated time points and the products separated and visualized as in D and E. (G) Quantification of the autoradiograph on the left using ImageJ software. For HJ, two cleavage rates were calculated: one by taking only the double stranded product into account (HJ-ds), and the other by taking both the double stranded and the single stranded products into account.</p

<i>slx-1</i> mutants exhibit hypersensitivity to nitrogen mustard, camptothecin, and ultraviolet C, but not ionizing radiation.

<p>Relative hatching frequencies detected in wild type, <i>slx-1, him-18</i> and <i>slx-1;him-18</i> mutants after treatment with the indicated doses of (A) ionizing radiation (IR), (B) nitrogen mustard (HN2), (C) camptothecin (CPT), and (D) ultraviolet C (UVC). Hatching is plotted as a fraction of the hatching observed in untreated animals. Error bars indicate standard error of the mean for ∼20 animals in each of three independent experiments. Colored asterisks indicate statistical significance between different genotypes.</p

SLX-1 promotes the regulation of crossover distribution but not crossover frequency.

<p>(A) Schematic representation of chromosome domains. Four SNP markers for chromosome III, IV and V and five SNP markers for the X chromosome are shown as A, B, B', C and D. The intervals A–B, B–C and C–D indicate portions of the left arm, center, and right arm, respectively. Purple lines indicate the central domain on which crossover frequency is lower compared to the arms defined by Rockman and Kruglyak <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002888#pgen.1002888-Rockman1" target="_blank">[79]</a>. (B) Analysis of crossover frequencies on chromosomes III, IV, V and X in wild type, <i>slx-1</i>, <i>him-18</i> and <i>slx-1;him-18</i> mutants. Crossover frequencies are measured between SNP positions A to D. N = number of crossprogeny scored. (C) Analysis of crossover distribution on chromosomes III, IV, V and X on the indicated genotypes. The positions on the chromosomes indicated as left, center and right correspond to intervals A–B, B–C and C–D, respectively as indicated on panel A. Asterisks indicate significant differences compared to wild type. (D) Crossover frequency of wild type and <i>slx-1</i> mutants between <i>dpy-10</i> and <i>unc-4</i> loci on center region of chromosome III. (E) Representative images of ZHP-3-GFP foci in late pachytene nuclei (zone 7) in wild type and <i>slx-1</i> mutants. The average number of ZHP-3-GFP foci is shown at the bottom left. Bars, 5 µm. (F) Quantitation of ZHP-3-GFP foci in late pachytene nuclei in wild type and <i>slx-1</i> mutants.</p

A model for how SLX-1 functions as a structure-specific endonuclease in various genome maintenance pathways.

<p>(A) Mitotic role of SLX-1. DNA repair pathways and the nuclease activity of SLX-1 are indicated. The HIM-18-dependent nuclease activity of SLX-1 is required for replication-coupled DNA repair such as during the re-establishment of a normal fork, DNA interstrand crosslink (ICL) repair, and nucleotide excision repair (NER). (A-i) The SLX-1-HIM-18 complex supports the RAD-51-mediated (green circles) repair pathway employed at replication forks. Specifically, SLX-1-HIM-18 resolves Holliday junctions when replication forks stall or collapse during S phase. Green scissors represent the SLX-1-HIM-18 nuclease complex. The cross represents a replication block. (A-ii) HIM-18-SLX-1 resolves HJs during re-establishment of a normal replication fork when CPT-induced single-ended DSBs are generated. (A-iii) The SLX-1-HIM-18 complex induces an incision at DNA ICLs when replication forks collide at an ICL. (A-iv) SLX-1-HIM-18 repairs UV-induced CPDs (orange triangles) via the replication coupled repair and NER pathways. Although it is thought that XPF and XPG make the 5′ and 3′ incisions, respectively, flanking the CPD lesion during the NER pathway in other organisms, there may be a possibility that SLX-1 also makes the 3′ incision, which is reminiscent of its 5′-flap cleavage activity. (B) SLX-1 is required for proper crossover distribution during meiotic recombination. Two alternative models are presented. (B-i) SLX-1 is a noncrossover specific Holliday junction resolvase. One of the evenly distributed DSBs are selected as a crossover precursor at one of the arm regions and marked by ZHP-3. DSBs located at the center of the chromosomes are repaired as noncrossovers via double Holliday junction resolution, synthesis-dependent strand annealing (SDSA) or intersister repair. SLX-1 might convert a dHJ into a noncrossover product by same sense dHJ resolution at the mid-section of chromosomes. (B-ii) Although the function of the PHD finger of SLX-1 is unknown, it is possible that a SLX-1-dependent modification or recognition of chromosome domains contributes to the regulation of crossover distribution.</p

The levels of H2AK5ac are similar between wild type and <i>slx-1</i> mutants.

<p>(A) Representative images of mid-pachytene nuclei stained with anti H2AK5ac in wild type, <i>slx-1</i> and <i>xnd-1</i> mutants. Bars, 5 µm. (B) Quantitation of H2AK5ac foci at mid pachytene in wild type, <i>slx-1</i> and <i>xnd-1</i> mutants. (C) Average number of H2AK5ac foci/nucleus in wild type, <i>slx-1</i> and <i>xnd-1</i> mutants. Bars indicate standard error.</p