BIOCHEMICAL ANALYSIS OF GENETIC RECOMBINATION IN YEAST

长期的目标是了解其分子机制。 真核生物中的同源重组。同源重组剧 在大多数生物体的生命周期中有两个重要的角色。它是 修复DNA中的致命损伤，如双链断裂， 它对于同源基因的配对和分离是必不可少的 减数分裂过程中的染色体。这些函数的重要性是 突变增加，有丝分裂和减数分裂的非整倍体 在没有重组的情况下。因为许多遗传性疾病是 与增加的基因组不稳定性相关，理解 重组的机制很可能对理解 这些疾病。此外，同源重组具有实用价值。 作为基因治疗工具的应用。我们的目标是确定 酵母中催化同源重组的蛋白质 使用遗传和生化方法的酿酒酵母。 酵母菌为这些研究提供了几个优势。它是一种相对的 简单的单细胞真核生物，很容易在有丝分裂中生长 单倍体或二倍体状态，可被诱导进行减数分裂 在简单液体介质中生长。它有一个定义明确的遗传系统 这使得分离出了一些改变了基因的突变体 重组。最后，酵母的完整DNA序列已经被 下定决心。大多数拟议的研究都集中在使用 菌落颜色扇形分析用于有丝分裂相关基因的筛选 重组。使用这项测试，我们已经证明了有丝分裂 RAD51的突变仅使重组减少了5倍，RAD51编码 细菌RecA蛋白的同源物。RAD59，它编码一个RAD52 Homolog，是根据其对RAD51非依赖性的要求而分离的 重组。RAD59蛋白将被提纯并鉴定 生化方面的。还将测试与Rad52的交互。其他基因 该功能在RAD51非依赖性重组途径中发挥作用，在 具体来说，涉及交换的基因将被用来识别 菌落分割试验。最后，我们计划对Mre11进行描述 吉恩。遗传和分子研究表明，Mre11是最重要的 处理双链末端的活动的候选人 休息一下。预计Mre11中的保守残基对 核酸酶活性将被改变，并进行体内功能测试，以及 Mre11蛋白复合体将被提纯，以检验这一假设 是一种核酸酶。

The long term objectives are to understand the molecular mechanisms of homologous recombination in eukaryotes. Homologous recombination plays two essential roles during the life cycle of most organisms. It is required to repair lethal lesions in DNA, 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 genetic diseases are associated with increased genome instability, an understanding of the mechanisms of recombination is likely to be important in understanding these diseases. Furthermore, homologous recombination has practical application as a tool for gene therapy. Our goals are to identify the proteins that catalyze homologous recombination in the yeast Saccharomyces cerevisiae using both genetic and biochemical approaches. Yeast offers several advantages for these studies. It is a relatively simple, unicellular eukaryote that is easily grown mitotically in a haploid or diploid state, and that can be induced to undergo meiosis by growth in simple liquid medium. It has a well-defined genetic system that has enabled the isolation of a number of mutants altered in recombination. Finally, the complete DNA sequence of yeast has been determined. Most of the proposed research is focused on the use of a colony color sectoring assay to identify genes involved in mitotic recombination. Using this assay we have shown that mitotic recombination is reduced only 5-fold by mutation of RAD51, which encodes a homolog of bacterial RecA proteins. RAD59, which encodes a Rad52 homolog, was isolated by its requirement for RAD51-independent recombination. The Rad59 protein will be purified and characterized biochemically. Interaction with Rad52 will also be tested. Other genes that function in the RAD51-independent recombination pathway, in particular, genes involved in crossing over, will be identified using the colony sectoring assay. Finally, we plan to characterize the MRE11 gene. Genetic and molecular studies indicate that Mre11 is the prime candidate for an activity that processes the ends of double-strand breaks. Conserved residues in Mre11 predicted to be important for nuclease activity will be altered and tested for in vivo function, and the Mre11 protein complex will be purified to test the hypothesis that is a nuclease.