The different pathways involved in meiotic recombination in mammals

虽然目前尚不清楚Mnd1基因敲除小鼠与Hop2小鼠是否会有本质上的不同，但现在已经清楚的是，这些小鼠是哺乳动物减数分裂的遗传信息宝库。与Hop2小鼠不同，Mnd1小鼠有相当大比例的精母细胞核显示出同源的成对染色体突触，修复所有双链断裂直到晚期粗线期，但不显示任何交叉。这些结果有力地表明，正如对发芽酵母所提出的那样，有两条主要途径(DSBR，双链断裂修复和SDSA，链置换和链退火)用于修复小鼠的双链断裂(DSBR主要导致交叉，SDSA只导致非交叉)，并且Hop2可以作用于这两条途径。这只小鼠还可能提供关于染色体相互作用主要是如何在同源基因与姐妹基因之间传递的洞察力，这是导致交叉的基本要求，确保染色体的适当分离。最近，我们已经证实，Hop2只在那些与染色体完全突触的Mnd1基因敲除的精母细胞中表达。这一发现强烈表明Hop2与这种突触有关，由于这些显示完全突触的精母细胞已经进入粗线期晚期，而正常情况下会出现交叉，这一发现也表明修复是通过SDSA发生的。我们还表明，尽管Hop2参与SDSA途径的研究是在Mnd1基因敲除的背景下进行的，但在野生型小鼠中，Hop2最有可能参与这一途径，因为在野生型精母细胞中，Hop2蛋白的数量是Mnd1蛋白的2到3倍，即Hop2的过剩超出了形成Hop2/Mnd1异源二聚体所需的数量，该异二聚体的功能是刺激RecA样重组酶、RAD51和Dmc1。 最近，我们发现在哺乳动物减数分裂过程中存在早期的DSB-和同源重组独立的染色体同源配对。减数分裂中同源染色体的配对和排列可以说是第一个遗传事件。我们已经提供了一些生化支持的流行观点(Yancey-Wrona和Camerini-Otero(1995)Current Biology 5,1149)认为，在从酵母到人类的大多数生物中，这种配对和比对是由SPO11引入的双链断裂(DSB)引发的全基因组同源性搜索的结果，并由同源重组(HR)生化机制介导。我们现在已经证明，在复制进入减数分裂(减数分裂S期)的小鼠生精细胞时，显著水平的配对先于染色体DNA的SPO11裂解。这些数据是通过在结构上保存的细胞核或组织切片中进行荧光原位杂交获得的，构成了哺乳动物早期配对的第一个报告。 使用一个缺乏SPO11催化活性的突变小鼠，我们已经证明了早期的染色体配对需要SPO11，但与它制造DSB的能力无关。这一发现与之前在发芽酵母中的观察结果是一致的(Cha等人。(2000)基因与发育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，在这一过程中不太可能需要HR。有趣的是，这种早期配对需要SUN1，一种参与端粒附着到核膜上的蛋白质(Ding X.等人。(2007)Dev Cell 12：863；迟勇等人。(2009)发育136：965)，对配子发生至关重要。此外，我们发现端粒上的DSB非依赖性配对是稳定的，而间质(非端粒)部位的配对是暂时的。我们提出，可能需要沿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入侵，这是在核苷酸水平上更精确和密切的染色体DNA比对的标志。也就是说，我们假设在减数分裂中，由DSB引发的同源重组实际上并不负责全基因组的同源搜索，而是校对初始配对以调节正常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，我们与东京大学渡边义史的实验室合作，研究了粘附素复合体在DSB前配对中的作用。对缺乏减数分裂粘附素复合体的小鼠在SPO11介导的切割之前配对的分析表明，它在早期配对中也发挥着重要作用，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基因敲除或其他转基因小鼠的方法来剖析早期减数分裂所涉及的途径。

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.