Joint Molecule formation during meiotic recombination

描述(由申请人提供)： 重组在人类减数分裂染色体分离中起着重要的机械作用，而缺陷重组与人类不孕、流产和遗传病有关。长期目标是了解重组的分子机制。减数分裂重组是通过DNA双链断裂(DSB)的程序化形成和处理发生的。DSB末端与同源染色体相互作用，首先形成单端侵袭(SEI)，然后形成双Holliday连接(DHJ)。具体的假设是，DSB到SEI和SEI到dHJ的转变通过不同的生化过程发生，Dmc1、RAD51和Rad52蛋白做出了独特的贡献。直接监测酿酒酵母细胞减数分裂重组的化学步骤的DNA物理分析将构成这项研究的基石。具体目的是：1.研究Dmc1和RAD51在体内的作用。零突变的间接影响以及缺乏体内检测相关链侵袭产物的方法，以前限制了我们对Dmc1和RAD51功能的理解。初步实验已经确定了当只有Dmc1或RAD51存在时减数分裂有效进行的条件。只有Dmc1和RAD51的重组将用物理、遗传学和细胞学分析来表征以下方面：(A)重组的DNA事件；(B)所涉及的基因产物；(C)形成的交叉的分布；(D)促进染色体配对和形成联会复合体的能力。2.研究SEI向dHJ转变的机制。SEI形成后的事件尚未确定，但必须包括第二个DSB末端的相互作用、DNA合成和连接。初步证据表明，第二个DSB末端可以通过单链退火过程相互作用。物理分析将被用来描述SEI到dHJ转变的以下两个方面：(A)通过分析一系列突变菌株来确定在单链退火过程中涉及的蛋白质的作用；(B)将使用DNA合成的化学抑制剂和复制蛋白质的条件等位基因来确定DNA合成的作用和涉及的因素。

DESCRIPTION (provided by applicant): Recombination plays an essential mechanical role in meiotic chromosome segregation and defective recombination is linked to infertility, miscarriage and genetic disease in humans. The long term goal is to understand the molecular mechanism of recombination. Meiotic recombination occurs by the programmed formation and processing of DNA double-strand breaks (DSBs). DSB-ends interact sequentially with an homologous chromosome forming first a Single-End Invasion (SEI) and then a double-Holliday Junction (dHJ). The specific hypothesis is that the DSB-to-SEI and SEI-to-dHJ transitions occur via biochemically distinct processes, with unique contributions being made by the Dmc1, Rad51 and Rad52 proteins. DNA physical assays to directly monitor the chemical steps of meiotic recombination in Saccharomyces cerevisiae cells will form the cornerstone of this investigation. The Specific Aims are: 1. To characterize the in vivo roles of Dmc1 and Rad51. Indirect effects of null mutations and lack of in vivo assays to detect relevant strand-invasion products have previously limited our understanding of Dmc1 and Rad51 function. Preliminary experiments have identified conditions in which meiosis progresses efficiently when only Dmc1 or Rad51 is present. The following aspects of Dmc1-only and Rad51-only recombination will be characterized using physical, genetic and cytological assays: (a) the DNA events of recombination; (b) the gene products involved; (c) the distribution of crossovers formed; (d) the ability to promote chromosome pairing and formation of synaptonemal complex. 2. To characterize the mechanism of the SEI-to-dHJ transition. Events following SEI formation are uncharacterized but must include interaction of the second DSB-end, DNA synthesis and ligation. Preliminary evidence suggests the second DSB-end can interact by a process of single-strand-annealing. Physical assays will be used to characterize the following two aspects of the SEI-to-dHJ transition: (a) the roles of proteins implicated in the process of single-strand-annealing will be determined by analyzing a series of mutant strains; (b) the role of DNA synthesis and the involved factors will be established using chemical inhibitors of DNA synthesis and conditional alleles of replication proteins.