MEIOTIC INTERACTIONS TO THE RECA HOMOLOGUE DMC1

在减数分裂过程中需要同源重组来促进第一次减数分裂时同源染色体的准确分离。已经确定了酵母减数分裂重组所需的几种蛋白质，但这些蛋白质在重组中的具体作用在很大程度上是未知的。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。本项目重点研究减数分裂重组所需的减数分裂蛋白Dmcl。Dmc1及其相关基因Rad51与RecA蛋白在结构和功能上具有同源性。RecA是大肠杆菌DNA损伤重组修复所需的中心蛋白。酵母Rad51，可能还有Dmc1，共享RecAs促进两个分子间链交换的能力。Dmcl蛋白将被纯化和表征，以确定其与RecA共享的活性。重组产物的形成除了RecA显示的那些酶活性外，还需要许多酶活性。减数分裂重组所需的许多蛋白质形成多蛋白质复合体。亲和色谱法和串联质谱法将用于鉴定在重组过程中与Dmcl形成复合物的蛋白质。时间过程和突变分析将用于描述功能重组复合物组装和拆卸的总体进展。此外，共聚焦显微镜将用于使用绿色荧光蛋白检查活细胞中的单个重组事件。Rad51和Dmc1的功能只是部分冗余。我们提出突变体筛选，旨在将Dmc1的冗余功能与其独特功能分离开来。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的功能由检查点控制机制监测，该机制确保在染色体分离发生之前重组完成。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中具有额外的功能。这些新确定的功能和检查点控制之间的关系将被检查。

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.