Mechanisms of Homologous Recombination in Yeast

这一研究计划的长期目标是了解同源基因的分子机制。 在真核生物模型中的重组，萌芽酵母酿酒酵母。同源 重组(HR)在大多数生物的生命周期中扮演着两个重要的角色。它需要修理 DNA的致命性损伤，如双链断裂，对于DNA的配对和分离是必不可少的 减数分裂过程中的同源染色体。这些功能的重要性从以下方面得到了证明： 突变，以及无重组的有丝分裂和减数分裂非整倍体。因为许多人类 癌症易发综合征与基因组不稳定性增加有关，了解 重组的机制可能对理解这些疾病很重要。同源 重组始于双链断裂时5‘-3’切除形成的单链DNA(SsDNA) (DSB)，或在停滞的复制分叉。RAD51催化单链DNA间的突触和链交换 结合在RAD51核蛋白细丝和同源双链DNA内，在需要RPA的反应中， RAD52、RAD54、RAD55和RAD57。对RAD51催化的链的理解已经取得了很大的进展 侵袭性，但切除核酸酶的同一性和侵袭后步骤所需的因素在很大程度上是 未知。我们已经开发了一种简单的单端链侵袭的基因检测方法，需要广泛的 DNA合成以完成重组修复。这个化验结果将被用来描述入侵后 人力资源使用物理监测方法的步骤，并确定涉及的复制因素。的作用 还将调查总染色体重排中的BIR。第二个目标是 切除核酸酶的鉴定。因为在断裂位点形成单链DNA被认为是一种 RAD51促进链侵袭的必备条件，令人惊讶的是没有RAD52组 基因编码切除核酸酶。这意味着这一步是冗余的，或者是切除 核酸酶是生存所必需的。人们提出了几种基因筛查来识别编码基因 核酸酶。最后，我们展示了高频R/AD57无关的重复组合 序列；建议进行遗传学研究以确定R/\D57独立重组的组成部分 路径。

The long-term objective of this research program is to understand the molecular mechanisms of homologous recombination in a model eukaryote, the budding yeast Saccharomyces cerevisiae. Homologous recombination (HR) plays two essential roles during the life cycle of most organisms. It is required to repair lethal lesions in DMA, such as double-strand breaks, and it is essential for the pairing and segregation of homologous chromosomes during meiosis. The importance of these functions is evidenced by increased mutagenesis, and mitotic and meiotic aneuploidy in the absence of recombination. Since many human cancer-prone syndromes are associated with increased genome instability, an understanding of the mechanisms of recombination is likely to be important in understanding these diseases. Homologous recombination initiates at single-stranded DNA (ssDNA) formed by 5'-3' resection at double-strand breaks (DSBs), or at stalled replication forks. Rad51 catalyzes synapsis and strand exchange between ssDNA bound within the Rad51 nucleoprotein filament and homologous duplex DNA, in a reaction requiring RPA, Rad52, Rad54, Rad55 and Rad57. Much progress has been made in understanding Rad51-catalyzedstrand invasion, but the identity of the resection nuclease and factors required for the post-invasion step are largely unknown. We have developed a simple genetic assay for one-ended strand invasion that requires extensive DNA synthesis to complete recombinational repair. This assay will be used to characterize the post-invasion steps of HR using physical monitoring methods and to identify the replication factors involved. The role of BIR in gross chromosomal rearrangements will also be investigated. The second aim is directed at identification of the resection nuclease. Because formation of ssDNA at break sites is considered an essential prerequisite for Rad51 -promoted strand invasion, it is surprising that none of the RAD52 group genes encodes the resection nuclease. This suggests redundancy for this step, or that the resection nuclease is essential for viability. Several genetic screens are proposed to identify genes encoding nucleases. Finally, we have shown high frequency R/AD57-independentrecombination of repeated sequences; genetic studies are proposed to identify components of the R/\D57-independent recombination pathway.