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

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.霍利迪连接体（Holliday junctions, HJs）是一类在重组事件中形成的十字形DNA结构。鉴定可促进这类结构解离的解离酶具有重要研究价值。本团队此前曾报道，秀丽隐杆线虫（Caenorhabditis elegans，以下简称C. elegans）的GEN-1是一种介导重组修复的对称切割型HJ解离酶，但未发现其在减数分裂重组中存在明显功能。本文中，我们鉴定了C. elegans中参与减数分裂HJ解离的蛋白。我们未发现GEN-1存在冗余的减数分裂功能。与之相反，我们发现了两条可能由SLX-4支架蛋白协同、且涉及HIM-6/BLM解旋酶的冗余HJ解离通路。 SLX-4可与SLX-1、MUS-81及XPF-1核酸酶结合，且此前被报道参与C. elegans的减数分裂重组。我们发现，C. elegans [mus-81; xpf-1]、[slx-1; xpf-1]、[mus-81; him-6]及[slx-1; him-6]双突变体的存活率均出现与slx-4突变体相似的下降。对减数分裂终变期染色体的分析显示，这类双突变体呈现独特的表型：野生型个体中的二价染色体消失，取而代之的是由染色质桥连接的成对"单价体"。这类连接依赖于保守的减数分裂特异性酯交换酶SPO-11，且可通过电离辐射修复，提示其代表未被解离的减数分裂HJ。上述结果表明存在两种主要的解离酶活性：一种由XPF-1与HIM-6提供，另一种由SLX-1与MUS-81提供。 在所有双突变体中，交换（crossover, CO）重组水平均出现下降但并未完全消失，提示减数分裂HJ解离过程中仍存在其他冗余机制。实时成像观察显示，第一次减数分裂过程中存在大量染色质桥，且这类结构最终似乎可在减数第二次分裂中被解离，表明存在于后期I或之后发挥作用的备用解离活性。我们还证实，在HJ解离突变体中，交换位点远端与近端的染色体臂仍可发生结构重塑，提示该过程仅需交换起始而非交换解离即可完成。