Meiotic Interactions of the RecA Homologue Dmc1 - Renewal 01

 描述（由申请方提供）：减数分裂对于健康配子（人类卵子和精子）的产生至关重要。分离在每条染色体的母本和父本拷贝之间产生物理连接，从而使它们在第一轮减数分裂染色体分离期间彼此分离成为可能。隔离的错误导致出生缺陷、流产和不孕。本项目主要研究减数分裂重组过程中关键酶Dmc 1的调控。Dmc 1在DNA双链断裂的位点组装成DNA-蛋白质细丝。这些丝状体能够在完整的染色单体中寻找与丝状体内结合的序列相匹配的序列，并且在发现了这样的序列之后，促进DNA单链的交换以形成杂交DNA的区域，所述杂交DNA的区域涉及来自母本染色体的DNA单链和来自父本染色体的互补DNA单链。 Dmc 1细丝的组装需要辅助因子的合作，包括称为Mei 5-Sae 3的异二聚体蛋白。在前一个研究周期中，我们发现Dmc 1的paraminase Rad 51（在有丝分裂细胞中提供唯一的链交换活性）被转化为辅助因子，与Mei 5-Sae 3一起发挥作用以刺激Dmc 1的活性。在这项工作中，我们将确定Rad 51和Mei 5-Sae 3合作的机制，使用传统的生物化学方法，结合使用超分辨率光学显微镜和其他显微镜方法的细丝结构的单分子分析。 一旦Dmc 1细丝组装，第二个辅助因子Hop 2-Mnd 1在体外刺激Dmc 1的活性约10至30倍。遗传实验还表明，Dmc 1的活性需要Hop 2-Mnd 1。在目前的资助期间，我们发现Hop 2-Mnd 1特异性刺激Dmc 1而不是Rad 51，从而提出Hop 2-Mnd 1负责Dmc 1在减数分裂中的专门功能。我们假设Hop 2-Mnd 1调节Dmc 1活性，使其优先促进与同源染色体而不是姐妹染色单体的重组，即重组是在母本和父本染色单体之间，而不是在两个母本或两个父本染色单体之间。在这里，我们提出了一个特定的分子模型来测试这种重组伴侣的偏见是如何实现的，通过调节染色体上的Hop 2-Mnd 1的分布。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（重组体）的结构。在之前的资助期间，我们发现证据表明Dmc 1在DNA双链断裂产生的DNA两端形成复合物，然后相互分离。我们建议确定这两个复合物是如何安排相对于蛋白质减数分裂特异性染色体支架结构称为轴向/横向元素。这些实验将揭示Dmc 1在基因组中搜索重组伴侣的机制。

 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.