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.
Highlights
Homologous recombination is important for error-free DNA double-strand break (DSB) repair and for meiotic crossover (CO) formation
Analysing a [mus-81 slx-1; xpf-1; him-6] quadruple mutant revealed a reduction in viability similar to that observed in the double mutants (Figure 1B)
We note that the reduction of viability observed in [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants equals the reduced viability observed in slx-4 mutants (Figure 1A, 1B)
Summary
Homologous recombination is important for error-free DNA double-strand break (DSB) repair and for meiotic crossover (CO) formation. Meiotic recombination is initiated by the introduction of programmed DSBs [1] by the conserved meiosis-specific Spo protein [2]. These DSBs are resected to produce 39 single-stranded DNA overhangs that, aided by RecA like recombinases (RAD-51 in C. elegans), initiate strand invasion into a homologous donor sequence [3]. Helicasedriven D-loop disassembly can occur, which in budding yeast is driven by the Sgs1/BLM-like helicase [14]. Such activities are ascribed to BLM in animals and further helicases such as RTEL are likely to play a similar role [15]. SDSA occurs relatively early during meiosis and appears to be set up independently of later RIs which can result in COs, at least in Author Summary
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