The different pathways involved in meiotic recombination in mammals

虽然目前还不清楚Mnd 1基因敲除小鼠是否与Hop 2小鼠有实质性的不同，但现在已经清楚的是，这些小鼠是哺乳动物减数分裂遗传信息的宝库。 与Hop 2小鼠不同，Mnd 1小鼠具有显着比例的精母细胞核，其显示同源配对的染色体突触，修复其所有双链断裂进展至粗线期晚期，但不显示任何交叉。这些结果强烈地表明，正如已经提出的芽殖酵母，有两个主要途径（DSBR，双链断裂修复和SDSA，链置换和链退火）用于修复小鼠中的双链断裂（DSBR主要导致交叉，SDSA仅导致非交叉），并且Hop 2可以作用于两个途径。这种小鼠也可能提供关于染色体相互作用主要是如何在同源物与姐妹篇之间传递的见解，这是导致确保染色体正确分离的交叉的基本要求。 最近，我们已经证实，Hop 2只表达在那些Mnd 1敲除精母细胞，突触他们的染色体完全。 这一发现强烈表明Hop 2负责这种突触，并且由于这些显示完整突触的精母细胞已经进行到粗线期后期的阶段，此时通常会出现交叉，这一发现也表明修复已经通过SDSA发生。 我们还表明，尽管在Mnd 1敲除背景中已经揭示了Hop 2参与SDSA途径，但在野生型小鼠中Hop 2最有可能参与该途径，因为在野生型精母细胞中Hop 2蛋白是Mnd 1蛋白的2至3倍，即，存在超过形成Hop 2/Mnd 1异源二聚体所需的过量Hop 2，所述异源二聚体起刺激RecA样重组酶Rad 51和Dmc 1的作用。 最近，我们已经建立了早期的DSB和同源重组独立的同源配对的染色体在哺乳动物减数分裂。减数分裂中同源染色体的配对和排列可以说是最重要的遗传事件。我们已经提供了一些生物化学支持（Yancey-Wrona and Camerini-Obern（1995）Current Biology 5，1149）的流行观点是，在从酵母到人的大多数生物体中，这种配对和比对是由SPO 11引入双链断裂（DSB）触发并由同源重组（HR）生物化学机制介导的全基因组同源性搜索引起的。我们现在已经表明，在复制小鼠生精细胞进入减数分裂（减数分裂S期）之前，染色体DNA的SPO 11切割的配对显着水平。这些数据，从荧光原位杂交在结构上保存的细胞核或组织切片，构成了这种早期配对在哺乳动物中的第一份报告。 使用一个突变小鼠缺乏催化活性的SPO 11，我们已经表明，早期染色体配对需要SPO 11，但独立于其能力，使DSBs。 这一发现与之前在芽殖酵母中的观察结果一致（Cha等人（2000）Genes and Development 14：493）。 此外，在缺乏几种HR蛋白的突变小鼠中对这种配对的检查证实，在这个过程中不太可能需要HR。有趣的是，这种早期配对需要SUN 1，一种参与端粒附着到核膜的蛋白质（丁X.等人（2007）Dev Cell 12：863; Chi Y.等人（2009）Development 136：965），并且对于配子发生是必需的。此外，我们发现，在端粒的DSB独立配对是稳定的，而在间质（非端粒）网站是短暂的。我们已经提出，可能需要沿着染色体长度的间隙配对的可逆性和瞬时性，以允许同源重组机制介导链侵入，这是染色体DNA在核苷酸水平上更精确和紧密对齐的标志。 也就是说，我们假设在减数分裂中，由DSB触发的同源重组实际上并不负责全基因组同源性搜索，而是校对初始配对以介导正确染色体突触的最后阶段。最后，我们现在正在研究这种早期同源配对的可能机制。在这方面，与东京大学Yoshi Watanabe的实验室合作，我们研究了cohesin复合物在Pre-DSB配对中的作用。在缺乏减数分裂粘附素复合体的小鼠中，对SPO 11介导的裂解之前的配对的分析表明，它在这种早期配对中也起着重要作用，类似于它在后期配对（突触）中的作用。 最近，我们一直在研究不依赖于使用基因敲除或其他转基因小鼠的方法来剖析参与早期减数分裂的途径。

Although it was not clear that Mnd1 knockout mice would be substantially different from Hop2 mice it has now become clear that these mice are a treasure trove of genetic information about mammalian meiosis. Unlike the Hop2 mice, the Mnd1 mice have a significant proportion of spermatocyte nuclei that show homologously paired chromosome synapsis,repair all their double-strand breaks progress up to late pachytene but do not show any crossovers. These results strongly suggest that, as has been proposed for budding yeast, there are two main pathways (DSBR, Double-Strand Break Repair and SDSA, Strand Displacement and Strand-Annealing) for the repair of double-strand breaks in mice (DSBR leads to mainly crossovers and SDSA results in non-crossovers exclusively) and that Hop2 can act on both pathways. This mouse might also provide insights into how chromosome interactions are channeled primarily between homologs versus sisters, a fundamental requirement leading to the crossovers that ensure the proper segregation of chromosomes. Recently, we have confirmed that Hop2 is only expressed in those Mnd1 knockout spermatocytes that synapse their chromosomes completely. This finding strongly suggests that Hop2 is responsible for this synapsis and since these spermatocytes that show complete synapsis have proceeded to a stage late in pachytene when crossovers would have normally appeared, this finding also indicates that the repair has occurred via the SDSA. We have also shown that although the involvement of Hop2 in the SDSA pathway has been revealed in the Mnd1 knockout background, Hop2 is most likely involved in this pathway in the wild-type mouse as there is 2 to 3 times as much Hop2 protein as there is Mnd1 protein in wild-type spermatocytes, that is, there is an excess of Hop2 beyond that required to form the Hop2/Mnd1 heterodimer that functions to stimulate the RecA-like recombinases, Rad51 and Dmc1. Recently, we have established that there is early DSB- and homologous recombination independent homologous pairing of chromosomes in mammalian meiosis. The pairing and alignment of homologous chromosomes in meiosis is arguably the premiere genetic event. The prevailing view, for which we have provided some biochemical support (Yancey-Wrona and Camerini-Otero (1995) Current Biology 5, 1149), is that in most organisms from yeast to man, this pairing and alignment results from a genome-wide search for homology triggered by the introduction of double-strand breaks (DSBs) by SPO11 and mediated by the homologous recombination (HR) biochemical machinery. We now have shown that a significant level of pairing in replicating mouse spermatogenic cells entering meiosis (meiotic S-phase) precedes SPO11 cleavage of chromosomal DNA. These data, obtained from fluorescent in situ hybridization in either structurally preserved nuclei or tissue sections, constitute the first report of such early pairing in mammals. Using a mutant mouse lacking the catalytic activity of SPO11, we have shown that early chromosome pairing requires SPO11, but is independent of its ability to make DSBs. This finding is consistent with previous observations in budding yeast (Cha et al. (2000) Genes and Development 14: 493). Furthermore, an examination of this pairing in mutant mice, deficient for several HR proteins confirmed that it is unlikely that HR is required in this process. Intriguingly, this early pairing requires SUN1, a protein involved in telomere attachment to the nuclear membrane (Ding X. et al. (2007) Dev Cell 12:863; Chi Y. et al. (2009) Development 136:965) and essential for gametogenesis. Furthermore, we find that the DSB-independent pairing at telomeres is stable while that at interstitial (non-telomeric) sites is transient. We have proposed that the reversibility and transience of the interstitial pairing along the length of the chromosomes may be required to allow the homologous recombination machinery to mediate the strand invasion that is the hallmark of the more precise and intimate alignment of the chromosomal DNA at the nucleotide level. That is, we posit that in meiosis, homologous recombination triggered by a DSB is not in fact, responsible for a genome-wide homology search but rather, proofreads the initial pairing to mediate the final stages of proper chromosomal synapsis. Finally, we are now investigating possible mechanisms for this early homologous pairing. In this regard, in a collaboration with the laboratory of Yoshi Watanabe at the University of Tokyo, we investigated the role of the cohesin complex in Pre-DSB pairing. Analysis of pairing prior to SPO11 mediated cleavage in mice lacking the meiotic cohesin complex shows that it also plays a significant role in this early pairing similar to its role in the late pairing (synapsis) later in prophase Most recently, we have been investigating approaches to dissect the pathways involved in early meiosis that do not rely on the use of gene knock-outs or other transgenic mice.