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.
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.
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DOI：
10.1371/journal.pgen.1003591
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Gartner A
中科院分区：
文献类型：
--
作者：
Agostinho A;Meier B;Sonneville R;Jagut M;Woglar A;Blow J;Jantsch V;Gartner A
Holliday连接(HJS)是在重组过程中产生的十字形DNA结构。确定促进这些结构分解的解析酶(S)是一件相当重要的事情。我们以前报道过线虫Gen-1是重组修复所必需的对称裂解HJ解析酶，但我们还没有发现在减数分裂重组中的一个明显的作用。在这里，我们鉴定了线虫参与解决减数分裂HJS的蛋白质。我们没有发现Gen-1有多余减数分裂功能的证据。相反，我们发现了两条冗余的HJ解析途径，可能由SLX-4支架蛋白协调，也涉及HIM-6/BLM解旋酶。SLX-4与SLX-1、MUS-81和XPF-1核酸酶相关，与线虫减数分裂重组有关。我们发现线虫[MUS-81；XPF-1]、[SLX-1；XPF-1]、[MUS-81；HIM-6]和[SLX-1；HIM-6]双突变株显示出与SLX-4相似的存活率下降。对减数分裂终变期染色体的分析表明，这两个突变体具有明显的表型。不是野生型的二价体染色体，而是由染色质桥连接的一对“单价体”。这些联系依赖于保守的减数分裂特异性转酯酶SPO-11，并可以通过电离辐射恢复，这表明它们代表未分解的减数分裂HJS。这表明存在两种主要的解旋酶活性，一种是由XPF-1和HIM-6提供的，另一种是由SLX-1和MUS-81提供的。在所有双突变体中，交叉(CO)重组减少，但不是取消，这表明减数分裂HJ分辨率进一步冗余。实时成像显示，在第一次减数分裂过程中，大量的染色质桥似乎最终在减数分裂II中被分解，这表明在后期I或之后发生的备份分辨活动。我们还表明，在HJ分辨突变体中，CO的远端和近端的染色体臂的重组仍然发生，这表明这一过程可能需要CO的启动而不是CO的分辨。减数分裂是一种特殊的细胞分裂，在一个S期之后是两轮细胞分裂，以确保单倍体配子的产生。减数分裂交叉(CO)重组是遗传多样性和第一次减数分裂过程中染色体正确分离所必需的。此外，在具有全着丝点染色体的生物中，COS是将减数分裂染色体分化为组织清晰的CO远端和CO近端区域所必需的。Holliday连接(HJS)是减数分裂重组过程中的关键中间产物，但我们对它们如何处理以确保CO重组知之甚少，特别是在动物中。在这项研究中，我们分析了线虫核酸酶和保守的HIM-6/BLM螺旋酶的结合需求，以调节减数分裂重组中间产物的加工。基于遗传和细胞学分析，我们认为HIM-6/BLM和XPF-1核酸酶，以及SLX-1和MUS-81核酸酶在促进HJ分解的两条多余途径中起作用。此外，我们提供的证据表明，对于减数分裂染色体向CO远端和CO近端区域的分化，CO起始而不是CO分解可能是重要的。
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. Meiosis is a specialized cell division, where a single S-phase is followed by two rounds of cell divisions to ensure the production of haploid gametes. Meiotic crossover (CO) recombination is required for genetic diversity and for the proper segregation of chromosomes during the first meiotic division. In addition, in organisms with holocentric chromosomes COs are required for differentiating meiotic chromosomes into distinctly organized CO distal and CO proximal domains. Holliday junctions (HJs) are crucial intermediates during meiotic recombination, but we still know very little about how they are processed to ensure CO recombination, especially in animals. In this study we analyse the combined requirement of C. elegans nucleases and the conserved HIM-6/BLM helicase to regulate the processing of meiotic recombination intermediates. Based on genetic and cytological analysis we propose that HIM-6/BLM and the XPF-1 nuclease, as well as the SLX-1 and MUS-81 nucleases act in two redundant pathways to promote HJ resolution. Furthermore, we provide evidence that CO initiation but not CO resolution is likely important for the differentiation of meiotic chromosomes into CO distal and CO proximal domains.
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