Joint Molecule formation during meiotic recombination

减数分裂重组过程中联合分子的形成

• 批准号: 6907652
• 负责人: NEIL HUNTER
• 金额: $ 24.86万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6907652
• 关键词: DNA; DNA binding protein; DNA replication; Saccharomyces cerevisiae; cell cycle proteins; chromosome movement; cytology; enzyme activity; fluorescent in situ hybridization; fungal genetics; fungal proteins; gene mutation; genetic recombination; genetic techniques; immunofluorescence technique; meiosis; molecular biology; molecular genetics; mutant; nuclear matrix; phenotype; protein protein interaction; protein structure function; recombinase

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.

描述（由申请人提供）： 减数分裂染色体分离和缺陷重组中起着重要的机械作用，与人类不育，流产和遗传疾病有关。长期目标是了解重组的分子机制。减数分裂重组通过DNA双链断裂（DSB）的程序化形成和加工而发生。DSB末端依次与同源染色体相互作用，首先形成单末端侵入（SEI），然后形成双霍利迪连接（dHJ）。具体的假设是，DSB到SEI和SEI到dHJ的转换通过生物化学上不同的过程发生，Dmc 1，Rad 51和Rad 52蛋白质做出了独特的贡献。DNA物理分析，直接监测化学步骤的减数分裂重组在酿酒酵母细胞将形成这项调查的基石。具体目标是：1。为了表征Dmc 1和Rad 51的体内作用。无效突变的间接影响和缺乏检测相关链侵入产物的体内测定限制了我们对Dmc 1和Rad 51功能的理解。初步实验已经确定了只有Dmc 1或Rad 51存在时减数分裂有效进行的条件。将使用物理、遗传和细胞学测定来表征仅Dmc 1和仅Rad 51重组的以下方面：（a）重组的DNA事件;（B）涉及的基因产物;（c）形成的交换的分布;（d）促进染色体配对和联会复合体形成的能力。2.表征SEI向dHJ转变的机制。SEI形成后的事件是未表征的，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。将使用物理测定来表征SEI至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单链退火过程中涉及的蛋白质的作用;（B）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。