GENE CONVERSION AND DNA MISMATCH CORRECTION IN YEAST

重组中间体中DNA碱基错配的校正 被认为是基因转化发生的主要途径。在……里面 在真核生物中，基因转换可能是维持基因 基因家族之间的稳定性。错配校正也是医学上的 兴趣，因为它有能力降低突变率；潜在的 单碱基改变激活细胞癌基因下划线 这一贡献。然而，真核生物中的错配纠正知识 是有限的。 这个项目的长期目标是一个详细的生化 酿酒酵母DNA错配校正特性的研究。 重点将放在PMS依赖途径上，因为这条途径是 已知会影响基因转换，这种影响与错配是一致的 修复功能，并已分离出经前综合征突变体。具体目标 是：1)表征可能影响的顺式作用因素 反应性，如单链断裂和不同的错配；2) 开发减数分裂细胞活性提取物并测试PMS依赖性；3) PMS1蛋白及其与大肠杆菌同源蛋白的分离和鉴定 MutS(失配识别)；以及4)确定其他所需因素和 开始隔离他们。 酵母菌错配校正的体外检测将基于 用于大肠杆菌系统的成功原型。异双链DNA 包含单个碱基错对的准备使得对 错配可以通过限制性内切酶裂解来监测。共价的 封闭的、有缺口的或有缺口的DNA将由酵母菌进行测试以进行校正 萃取物，以及含有不同成分的杂双链的反应性 单碱基错配。反应的效率和滞后性 将对这些修改进行分析。萃取物将从 诱导细胞进行减数分裂，预期活性可能是 在这些条件下得到了增强。这项活动对 Pms1、pms2和pms3将通过检测菌株的提取物来确定。 缺乏这些基因产物。PMS1蛋白将被过度生产并 通过检测缺陷提取物的补充性而分离出来的。一种蛋白质 将被过度生产并通过分析补充 有缺陷的摘录。还将分离出一种与MutS同源的蛋白质。 将对这些因素进行测试，以确定预计将涉及的活动 例如错配识别或核酸酶功能的纠正。其他 所需的蛋白质将通过筛选菌株的提取液来鉴定 缺乏DNA新陈代谢(如DNA聚合)，导致不匹配 修正活动。涉及的蛋白质将被分离出来。一位直接的 还将进行错配修复突变体的选择。

The correction of mismatched DNA bases within recombination intermediates is thought to be a major pathway by which gene conversion occurs. In eukaryotes, gene conversion may be a key mechanism for maintaining genetic stability among gene families. Mismatch correction is also of medical interest by virtue of its ability to reduce mutation rates; the potential for activation of cellular oncogenes by single base changes underscores this contribution. However, knowledge of mismatch correction in eukaryotes is limited. The long-term goal of this project is a detailed biochemical characterization of DNA mismatch correction in the yeast S. cerevisiae. The focus will be on the pms- dependent pathway because this pathway is known to affect gene conversion, the effects are consistent with mismatch repair function, and pms mutants have been isolated. The specific aims are: 1) to characterize cis-acting factors that are likely to affect reactivity, such as single-strand breaks and different mispairs; 2) to develop active extracts of meiotic cells and test pms-dependence; 3) to isolate and characterize PMS1 protein and a protein homologous to E. coli MutS (mismatch recognition); and 4) to identify other required factors and begin their isolation. An in vitro assay for yeast mismatch correction will be based on the successful prototype used for the E. coli system. Heteroduplex DNA containing a single base mispair is prepared such that correction of the mismatch can be monitored by restriction enzyme cleavage. Covalently closed, nicked or gapped DNA will be tested for correction by yeast extracts, as will the reactivity of heteroduplexes containing different single base mispairs. The efficiency and strandedness of the reaction due to these modifications will be analyzed. Extracts will be prepared from cells induced for meiosis with the expectation that activity may be enhanced under these conditions. The dependence of this activity on pms1,pms2, and pms3 will be determined by assaying extracts from strains deficient in these gene products. PMS1 protein will be overproduced and isolated by assaying for complementation of defective extracts. A protein will be overproduced and isolated by assaying for complementation of defective extracts. A protein homologous to MutS will also be isolated. These factors will be tested for activities expected to be involved in correction such as mispair recognition or nuclease function. Other required proteins will be identified by screening extracts of strains deficient in DNA metabolism (e.g. DNA polymerization) for mismatch correction activity. The proteins involved will be isolated. A direct selection of mismatch repair mutants will also be performed.