MOLECULAR MECHANISMS OF GENETIC RECOMBINATION IN YEAST

该项目的长期目标是了解分子 减数分裂重组的机制和联会丝的作用 复合物（SC）。正确的染色体必须进行纯化 在减数分裂的第一次分裂分离，因此， 用于在有性生殖中产生可存活的整倍体配子 有机体我们已经确定了S。酿酒酵母突变体缺陷型 染色体联会和减数分裂重组。未来的研究 预计突变体可以鉴定出酶和中间体， 重组，并提供深入了解的结构和功能， SC。 ZIP1基因编码SC组分。我们将研究的影响， zip1突变对减数分裂重组、交叉干扰和减数分裂的影响 染色体分离zip1的多拷贝和第二位点抑制子 将分离突变以鉴定ZIP1相互作用蛋白。 我们将克隆和表征在2010年的研究中， 减数分裂响应zip1，我们将物理表征 在zip1细胞中积累的重组中间体。ZIP1将是 位于SC内，并且苍白，但无突触， 将研究在Zip1菌株中观察到的同源物。 rec10突变体表现出一种新的表型：染色体突触，但 减数分裂重组不被诱导，表明重组是 突触不需要。我们将确定rec10菌株是否 减数分裂双链断裂（DSB）。REC10蛋白质的功能包括： 有丝分裂和减数分裂和免疫定位表明，REC 10是一个 染色质的组成部分。我们建议研究rec10对 减数分裂基因表达、染色质结构和细胞周期进程。 将检查REC10基因表达以鉴定有丝分裂和减数分裂。 特定的转录物和蛋白质。 以前对CON 5和CON 6的研究表明，这些基因的产物 直接参与重组的酶学过程。我们将 确定con5和con6突变对基因转换的影响 频率和束长度，并对DSB的形成，异源双链DNA 将对CON 5和CON 6基因进行克隆、测序和破坏 并且编码的蛋白质将被免疫定位。 许多基因的突变体不能经历减数分裂诱导 重组或使SC。我们建议确定mei4， mer2和rec102突变体产生减数分裂DSB，（如果不是） 可以通过人工诱导的DSB绕过重组缺陷。在 此外，我们将确定是否同源染色体配对原位 杂交使减数分裂染色体扩散。 msh4突变体减少相互交换，但不减少基因转换。 我们将在减数分裂细胞内定位MSH4蛋白，并检测MSH4 蛋白质在体外结合霍利迪连接的能力。

The long term goal of this project is to understand the molecular mechanism of meiotic recombination and the role of the synaptonemal complex (SC). Recombination is essential for correct chromosome segregation at the first division of meiosis and is, therefore, required for the production of viable euploid gametes in sexually reproducing organisms. We have identified S. cerevisiae mutants defective in chromosome synapsis and meiotic recombination. Future studies of these mutants are expected to identify enzymes and intermediates in recombination and to provide insight into the structure and function of the SC. The ZIP1 gene encodes an SC component. We will examine the effect of the zip1 mutation on meiotic recombination, crossover interference and meiotic chromosome segregation. Multicopy and second-site suppressors of zip1 mutations will be isolated in order to identify ZIP1-interacting proteins. We will clone and characterize checkpoint genes required for the arrest in meiosis in response to zip1 and we will physically characterize the recombination intermediate that accumulates in zip1 cells. ZIP1 will be localized within the SC and the connections between paled, but unsynapsed, homologs observed in zip1 strains will be investigated. The rec10 mutant displays a novel phenotype; chromosomes synapse, but meiotic recombination is not induced, demonstrating that recombination is not required for synapsis. We will determine whether rec10 strains make meiotic double-strand breaks (DSBs). The REC10 protein functions both in mitosis and in meiosis and immunolocalization suggests that REC10 is a component of chromatin. We propose to examine the effect of rec10 on meiotic gene expression, chromatin structure and cell cycle progression. REC10 gene expression will be examined to identify mitosis- and meiosis- specific transcripts and proteins. Previous studies of CON5 and CON6 suggest that the products of these genes are directly involved in the enzymology of recombination. We will determine the effect of the con5 and con6 mutations on gene conversion frequency and tract length, and on the formation of DSBs, heteroduplex DNA and SC. The CON5 and CON6 genes will be cloned, sequenced and disrupted and the encoded proteins will be immunolocalized. Mutants in a number of genes fail to undergo meiotically-induced recombination or to make SC. We propose to determine whether the mei4, mer2 and rec102 mutants make meiotic DSBs and (if not) whether the recombination defect can be bypassed an artificially induced DSB. In addition, we will determine whether homologous chromosomes pair by in situ hybridization to spread meiotic chromosomes. The msh4 mutant reduces reciprocal crossing over, but not gene conversion. We will localize the MSH4 protein within meiotic cells and test MSH4 protein for the ability to bind Holliday junctions in vitro.