MEIOTIC INTERACTIONS OF THE RECA HOMOLOGUE DMCL

RECA 同源物 DMCL 的减数分裂相互作用

• 批准号: 2853615
• 负责人: DOUGLAS K BISHOP
• 金额: $ 28万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2853615
• 关键词: DNA repair; Saccharomyces cerevisiae; affinity chromatography; cell cycle proteins; confocal scanning microscopy; fungal proteins; gene mutation; genetic recombination; green fluorescent proteins; mass spectrometry; meiosis; protein purification; protein structure function

Homologous recombination is required during meiosis to promote accurate segregation of homologous chromosomes at the first meiotic division. Several proteins have been identified that re required for MEIOTIC RECOMBINATION in the YEAST Saccharomyces, but the specific role of these proteins in recombination is largely unknown. The long term goals of this work are to determine the molecular mechanism of meiotic recombination and how the cell cycle machinery coordinates recombination with other meiotic events. This project focuses on one meiotic protein, Dmcl, that is required for meiotic recombination. Dmc1 and its relative Rad51 share structural and functional homology to RecA protein. RecA is the central protein required fo recombinogenic REPAIR OF DNA DAMAGE in E. Coli. Yeast Rad51, and probably Dmc1 as well, share RecAs ability to promote strand exchange between two molecules. Dmcl protein will be purified and characterized to determine what activities it shares with RecA. The formation of recombination products requires numerous enzymatic activities in addition to those displayed by RecA. Many of the proteins required for meiotic recombination form a multi protein complex. Affinity chromatography and tandem mass spectroscopy will be used to identify proteins that form complexes with Dmcl during recombination. Time-course and mutational analyses will be used to characterize overall progress in the assembly and disassembly of functional recombination complexes. In addition, confocal microscopy will be used to examine individual recombination events in living cells using green fluorescent protein. The functions of Rad51 and Dmc1 are only partially redundant. We propose mutant screens designed to separate the redundant function of Dmc1 from its unique functions. These mutations will then be used to characterize the mechanistic features of meiotic recombination that distinguish it from mitotic recombinational repair. The function of Dmc1 is monitored by a checkpoint control mechanism that ensures recombination is complete before chromosome segregation occurs. We recently showed the genes required for checkpoint control have additional functions in synapsis and suppression of ectopic recombination. The relationship between these newly identified functions and checkpoint control will be examined.

在减数分裂过程中需要同源重组，以促进第一次减数分裂时同源染色体的准确分离。酵母减数分裂重组所需的几种蛋白质已被鉴定，但这些蛋白质在重组中的具体作用尚不清楚。这项工作的长期目标是确定减数分裂重组的分子机制，以及细胞周期机制如何协调重组与其他减数分裂事件。该项目主要研究减数分裂重组所需的一种减数分裂蛋白Dmc1。Dmc1及其相关的RAD51与RecA蛋白在结构和功能上具有同源性。RecA是重组基因修复DNA损伤所必需的中心蛋白。酵母RAD51，可能还有Dmc1，也分享了Recas促进两个分子之间链交换的能力。将对DmCL蛋白进行纯化和鉴定，以确定它与RecA有什么共同的活性。重组产物的形成除了RecA所显示的活性外，还需要许多酶活性。减数分裂重组所需的许多蛋白质形成一个多蛋白质复合体。亲和层析和串联质谱学将用于鉴定在重组过程中与DmCL形成复合体的蛋白质。时间进程和突变分析将被用来描述功能重组复合体组装和拆解的总体进展。此外，共聚焦显微镜将用于检查使用绿色荧光蛋白的活细胞中的个体重组事件。RAD51和DMC1的功能只是部分冗余。我们提出了突变筛选，旨在将Dmc1的冗余功能与其独特功能分开。这些突变将被用来描述减数分裂重组的机制特征，以区别于有丝分裂重组修复。Dmc1的功能受到检查点控制机制的监控，该机制确保重组在染色体分离发生之前完成。我们最近发现，检查点控制所需的基因在突触和抑制异位重组方面具有额外的功能。将审查这些新确定的功能与检查站控制之间的关系。