Meiotic Interactions of the RecA Homologue Dmc1 - Renewal 01

 描述(申请人提供)：减数分裂重组是产生健康配子(人类的卵子和精子)所必需的。重组在每条染色体的母本和父本之间建立物理连接，从而使它们有可能在第一轮减数分裂染色体分离过程中彼此分离。错误的隔离会导致出生缺陷、流产和不孕不育。该项目针对减数分裂重组Dmc1中的关键中心酶的调节。Dmc1在DNA双链断裂处组装成DNA-蛋白质细丝。这些细丝能够搜索完整的染色单体，寻找与细丝内结合的序列相匹配的序列，并在找到这样的序列后，促进DNA单链的交换，形成杂交DNA的区域，涉及来自母体染色体的DNA单链和来自父本染色体的互补单链。Dmc1细丝的组装需要辅助因子的合作，其中包括一种名为Mei5-Sae3的异源二聚体蛋白质。在上一个授予周期中，我们发现在有丝分裂细胞中提供唯一链交换活性的Dmc1‘S旁系RAD51被转化为一个辅助因子，与Mei5-SAE3一起作用于刺激Dmc1’S的活性。在这项工作中，我们将使用传统的生化方法结合超分辨光学显微镜和其他微观方法的单分子细丝结构分析来确定RAD51和MEI5-SAE3相互作用的机制。一旦Dmc1纤维被组装，第二个辅助因子Hop2-Mnd1在体外可以刺激Dmc1的活性大约10到30倍。基因实验还表明，Dmc1的S活性需要Hop2-Mnd1。在目前的资助期间，我们发现Hop2-Mnd1特异性地刺激Dmc1，而不是RAD51，导致提出Hop2-Mnd1负责Dmc1在减数分裂中的特殊功能。我们假设Hop2-Mnd1调节Dmc1的活性，从而优先促进与同源染色体而不是姐妹染色单体的重组，即重组发生在母亲和父亲的染色单体之间，而不是两个母亲或父亲的染色单体之间。在这里，我们建议测试一个特定的分子模型，说明这种重组伙伴偏见是如何通过Hop2-Mnd1在染色体上的调节分布来实现的。最后，我们将使用超高分辨率光学显微镜来表征减数分裂重组复合体(重组小体)的结构。在之前的资助期间，我们发现了Dmc1在DNA两端形成复合体的证据，这些复合体是由DNA双链断裂产生的，然后相互分离。我们建议确定这两个复合体相对于蛋白质减数分裂特定的染色体支架结构(称为轴/侧元件)是如何排列的。这些实验将揭示Dmc1在基因组中寻找重组伙伴的机制。

 DESCRIPTION (provided by applicant): Meiotic Recombination is essential for the production of healthy gametes (egg and sperm in humans). Recombination creates physical connections between the maternal and paternal copies of each chromosome thereby making it possible for them to disjoin from one another during the first round of meiotic chromosome segregation. Errors in segregation result in birth defects, miscarriages, and infertility. This project is direced at the regulation of the key central enzyme in meiotic recombination Dmc1. Dmc1 is assembled into DNA-protein filaments at sites of DNA double strand breaks. These filaments have the ability to search intact chromatids for sequences that match the sequence bound within the filament and, having found such sequences, promote an exchange of DNA single strands to form regions of hybrid DNA involving a DNA single strand from the maternal chromosome and the complementary single DNA strand from the paternal chromosome. Assembly of Dmc1 filaments requires cooperation of accessory factors including a heterodimeric protein called Mei5-Sae3. In the previous grant cycle we showed that Dmc1's paralogue Rad51, which provides the only strand exchange activity in mitotic cells, is converted to an accessory factor that functions with Mei5-Sae3 to stimulate Dmc1's activity. In this work we will determine the mechanism through which Rad51 and Mei5-Sae3 cooperate using traditional biochemical approaches combined with single molecule analysis of filament structure using super-resolution light microscopy and other microscopic methods. Once Dmc1 filaments are assembled, a second accessory factor, Hop2-Mnd1 stimulates the activity of Dmc1 about 10 to 30 fold in vitro. Genetic experiments also show that Dmc1's activity requires Hop2-Mnd1. During the current funding period we showed that Hop2-Mnd1 specifically stimulates Dmc1 and not Rad51 leading to the proposal that Hop2-Mnd1 is responsible for the specialized function of Dmc1 in meiosis. We hypothesize that Hop2-Mnd1 regulates Dmc1 activity such that it preferentially promotes recombination with a homologous chromosome rather than a sister chromatid, i.e. such that recombination is between a maternal and paternal chromatid rather than between two maternal or two paternal chromatids. Here we proposed to test a specific molecular model for how this recombination partner bias is achieved via the regulated distribution of Hop2-Mnd1 on chromosomes. Finally, we will use super-high resolution light microscopy to characterize the structure of the meiotic recombination complex (recombinosome). In the previous funding period we found evidence that Dmc1 is forms complexes on both DNA ends created by a DNA double strand break which then separate from one another. We propose to determine how the two complexes are arranged with respect to the proteinaceous meiosis-specific chromosome scaffold structure called the axial/lateral element. These experiments will shed light the mechanism through which Dmc1 searches the genome for recombination partners.