GENE CONVERSION AND DNA MISMATCH CORRECTION IN YEAST

重组中间体中错配DNA碱基的校正 被认为是基因转换发生的主要途径。 在 真核生物中，基因转换可能是维持遗传的关键机制。 基因家族的稳定性。失配校正也是医学上的 由于其降低突变率的能力， 通过单碱基变化激活细胞癌基因的研究强调 这一贡献。 然而，真核生物中的错配校正知识 是有限的。 这个项目的长期目标是一个详细的生物化学 酵母S.啤酒。 重点将放在经前综合症依赖的途径上，因为这一途径是 已知影响基因转换，其影响与错配一致 修复功能和PMS突变体已经被分离出来。 具体目标 是：1）表征可能影响的顺式作用因子 反应性，如单链断裂和不同的错配; 2） 开发减数分裂细胞的活性提取物并测试PMS依赖性; 3） 分离并鉴定PMS 1蛋白和与E.杆菌 MutS（错配识别）;以及4）识别其他所需因素， 开始隔离。 酵母错配校正的体外试验将基于 成功的原型用于E. coli系统。 异源双链DNA 制备含有单个碱基错配的寡核苷酸序列，使得所述错配的校正 错配可以通过限制酶切割来监测。 共价 封闭的、有缺口的或有缺口的DNA将被酵母菌检测以进行校正 提取物，以及含有不同的异源双链体的反应性 单碱基错配 反应的效率和链化程度， 将对这些修改进行分析。 浸提液将由 诱导减数分裂的细胞，预期活性可能是 在这种条件下得到加强。 这种活动对 pms 1、pms 2和pms 3将通过测定菌株的提取物来确定 缺乏这些基因产物。 PMS 1蛋白会过量产生， 通过测定缺陷提取物的互补作用来分离。 蛋白质 将被过量生产，并通过测定 有缺陷的提取物。 还将分离与MutS同源的蛋白质。 这些因素将在预期涉及的活动中进行测试， 例如错配识别或核酸酶功能。 其他 所需的蛋白质将通过筛选菌株提取物来鉴定 DNA代谢缺陷（如DNA聚合）导致错配 更正活动。 将分离所涉及的蛋白质。 直接 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.