Combinatorial Regulation of Meiotic Holliday Junction Resolution in C. elegans by HIM-6 (BLM) Helicase, SLX-4, and the SLX-1, MUS-81 and XPF-1 Nucleases

Abstract

Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of “univalents” linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation but not resolution is likely to be required for this process.

Crossover Heterogeneity in the Absence of Hotspots inCaenorhabditis elegans

“…In yeast, BGC is actually restricted to crossover-associated gene conversion tracts (Lesecque et al 2013), but the relationship between BGC and recombination pathway clearly evolves and varies among species (Capra and Pollard 2011;Poh et al 2012). Recent dissection of the partially redundant Holiday junction resolvases in C. elegans (Saito et al 2013;O'Neil et al 2013;Agostinho et al 2013) leads us to the hypothesis that slx-1, which is disproportionately responsible for noncrossover gene conversion in chromosome centers (Saito et al 2013), may be more prone to BGC than the other resolvases. BGC is expected to have very weak effects on equilibrium GC content in selfing organisms (Marais et al 2004), due to the scarcity of heterozygotes, but it could persist in C. elegans from evolution in its outcrossing ancestor (Cutter et al 2008).…”

Increased Meiotic Crossovers and Reduced Genome Stability in Absence of Schizosaccharomyces pombe Rad16 (XPF)

“…The role of XPF in meiosis varies considerably in different species. In Drosophila, Mei9 XPF is essential for resolution of meiotic chiasmata (Yildiz et al 2002), while in C. elegans, XPF overlaps with two other nucleases, Mus81 and SLX-1, in meiosis (Agostinho et al 2013;O'Neil et al 2013;Saito et al 2013). In budding yeast, Rad1 XPF appears to have no function in meiosis (Higgins et al 1983).…”

“…In Drosophila, the DmMei9 XPF homolog functions as a Holliday junction resolvase during meiosis; flies deficient in mei9 show reduced meiotic recombination and loss of viabile progeny (Yildiz et al 2002). In Caenorhabditis elegans, XPF functions redundantly with other structure-specific endonucleases MUS-81 and SLX-1 in resolution of crossovers, and suppresses formation of abnormal structures (Agostinho et al 2013;O'Neil et al 2013;Saito et al 2013). In humans and in budding yeast, the structure-specific endonucleases SLX1/Slx1, MUS81/Mus81, and GEN1/Yen1, but not XPF, are linked to crossover resolution of Holliday junctions (Kaliraman et al 2001;Fricke and Brill 2003;Wyatt et al 2013).…”

Meiotic Recombination: The Essence of Heredity

“…The crossover function of Drosophila SLX4 ortholog MUS312 involves not SLX1, but the XPF-ERCC1-family nuclease, MEI9 -ERCC1, which is responsible for the vast majority of crossovers in this organism (Andersen et al 2009). In C. elegans, XPF-1 also promotes crossing-over, functioning in parallel with a second pathway defined by MUS81 and SLX1 (Agostinho et al 2013;Bellendir and Sekelsky 2013;O'Neil et al 2013;Saito et al 2013). However, even when both pathways are mutated, !50% -70% of normal crossover levels can still form indicating the existence of a third major pathway of joint molecule resolution in C. elegans meiosis.…”