BIOCHEMICAL ANALYSIS OF GENETIC RECOMBINATION IN YEAST

我们的长期目标是了解其分子机制。 真核生物中的同源重组。同源重组发挥了两种作用 在MOST的生命周期中，重要但似乎相互矛盾的角色 有机体。一方面，重要的是通过创造 新的基因组合，或部分基因。从另一方面来说，它是 对于有丝分裂细胞中的DNA损伤的忠实修复，以及对 减数分裂过程中染色体的分离。后者的重要性 功能的证据是增加的突变，有丝分裂和减数分裂 缺乏重组功能的非整倍体。因为许多遗传基因 疾病与基因组的不稳定性增加有关， 理解重组的机制可能是很重要的。 在理解这些疾病方面。我们选择了用酵母 酿酒酵母作为这些研究的模型系统，因为它 很容易在基因和生物化学上进行操作，此外，还显示出 高频有丝分裂和减数分裂重组。这样做的主要目标是 建议提纯催化重组的蛋白质并鉴定 编码它们的基因。有证据表明，5英尺-3英寸 处理双链断裂位点需要核酸外切酶的活性 在斯特兰德入侵之前。我们已经鉴定了几种5‘-3’核酸外切酶 有丝分裂提取物中的活性。我们计划提纯其中的一个，并使用 纯化的蛋白质用于鉴定编码核酸酶的基因。突变者 将被构造以确定其在修复和修复中的角色 重组。我们已经建立了一种制备8型DNA的程序 含有Holliday连接的分子。用这种DNA作为底物 我们已经确定了一个解决Holliday连接的活动。这 活性将被提纯，其细胞功能将用 反向遗传学。Figure-8底物还将用于识别和 净化分支机构迁徙促进活动。除了这些之外， 生化研究，我们计划利用一个敏感的菌落分区 筛选减少自发有丝分裂重组的突变体。 我们分离更多突变体的目标是更好地理解 重组的基因控制，并增加可用的池 筛选生化活动中的缺陷。

Our long term objectives are to understand the molecular mechanisms of homologous recombination in eukaryotes. Homologous recombination plays two important, but seemingly contradictory roles in the life cycle of most organisms. On one hand it is important to generate diversity by creating new combination of genes, or parts of genes. On the other hand it is required for the faithful repair of DNA lesions in mitotic cells, and for segregation of chromosomes during meiosis. The importance of the latter functions is evidenced by increased mutagenesis, and mitotic and meiotic aneuploidy in the absence of recombination functions. Since many genetic diseases are associated with increased genome instability, an understanding of the mechanisms of recombination is likely to be important in understanding these diseases. We have chosen to use the yeast Saccharomyces cerevisiae as a model system for these studies since it is easily manipulated genetically and biochemically and, in addition, shows high frequency mitotic and meiotic recombination. The primary goal of this proposal is to purify proteins that catalyze recombination and to identify the genes that encode them. Evidence has accumulated that 5'-3' exonuclease activity is required to process double-strand break sites prior to strand invasion. We have identified several 5'-3' exonuclease activities in mitotic extracts. We plan to purify one of these and to use the purified protein to identify the gene encoding the nuclease. Mutants will then be constructed to determine its role in repair and recombination. We have established a procedure for preparing figure-8 DNA molecules that contain a Holliday junction. Using such DNA as a substrate we have identified an activity that resolves Holliday junctions. This activity will be purified and its cellular function determined using reverse genetics. The figure-8 substrate will also be used to identify and purify branch migration promoting activities. In addition to these biochemical studies, we plan to utilize a sensitive colony sectoring screen to isolate mutants that decrease spontaneous mitotic recombination. Our goal in the isolation of more mutants is to better understand the genetic control of recombination, and to increase the available pool for screening for defects in biochemical activities.