ANALYSIS OF YEAST DNA REPAIR GENES RAD51, 52, AND 54

包括姐妹染色单体交换在内的染色体断裂修复是 酵母中的三种主要修复途径， X射线诱导的双链断裂，也参与修复 包括DNA交联在内的多种化学损伤。 哺乳动物细胞中可能也利用了免疫修复， 最近，已经分离出与这三种基因同源的人类基因。 我们将要研究的酵母基因。由于一些修复基因已经被 与致癌作用密切相关，更好地了解如何 修复基因的功能应该给我们重要的信息， 对致癌作用和细胞如何处理 暴露于环境致癌物。重组调查 修复酵母S.酿酒酵母，强调三个基因（RAD 51， 52和54）这一进程最必要的，将继续下去。的 具体目标与《区域发展议程》不同领域的重要性有关54 蛋白质，包括突变的蛋白质在内的大蛋白质家族的成员 在Cockayne综合征B中， 重组修复，以及基因在此过程中的功能顺序 通路拟议中的实验将为我们提供更多的信息 这个重要的修复途径。 RAD 54基因是重组修复所必需的，但它不是 广泛研究。将启动分子和生物化学研究， 检查编码推定的核苷酸结合的感兴趣的区域 结构域，潜在的DNA解旋酶结构域，和一个可能的锌指。网站 使用PCR引物的特异性诱变将用于引入 突变成潜在的域。这些突变将被测试， 对修复的影响低保真PCR将用于分离随机 rad 54突变，将筛选温度条件和 渗漏等位基因感兴趣的等位基因将被测序并用于其他研究。 实验结合这些分子实验，我们将 还分离了RAD 54蛋白和含有锌的截短形式， 手指区我们将尝试从E.大肠杆菌，但使用 在酵母中过量表达。这种蛋白质将被表征为 几种活性，包括ATP酶活性，锌结合，结合 链断裂和解旋酶活性。一些突变的RAD 54蛋白将 也被定性。 RAD 52和RAD 51蛋白最近被报道相互作用。- 我们 计划确定这种蛋白质相互作用是否对于 重组修复，通过进一步表征我们的rad 52 -20等位基因， 其在这种相互作用中可能是有缺陷的。为了测试rad 52 -20是否 蛋白质与RAD 51的相互作用减弱，我们将使用分子 双混合动力系统还将开展研究，以确定 RAD 54蛋白与其他蛋白相互作用，使用遗传逆转 条件性rad 54等位基因和双杂交系统。 实验提出了检查的功能顺序的RAD 51， 52和54个基因，使用温度变化实验结合 温度敏感的和冷敏感（c.s.）等位基因我们计划 为了确定RAD 54是在RAD 51和RAD 52之前还是之后工作，使用 我们最近隔离的c.s.和T. S. rad 51等位基因与 T.S. rad 54 -3等位基因和新的t.s. rad 52等位基因，由Kaytor分离， 利文斯顿。

Recombinational repair, including sister-chromatid exchange, is one of the three major repair pathways in yeast, and is crucial for the repair of x-ray induced double-strand breaks and is also involved in the repair of many types of chemical damage, including DNA cross-links. Recombinational repair is probably also utilized in mammalian cells, and recently human genes have been isolated which are homologs of the three yeast genes we will be studying. Since several repair genes have been strongly implicated in carcinogenesis, a better understanding of how repair genes function should give us important information which will have implications for both carcinogenesis and how cells deal with exposure to environmental carcinogens. Investigation of recombinational repair in the yeast S. cerevisiae, emphasizing the three genes (RAD51, 52 and 54) most necessary for this process, will be continued. The specific aims relate to the importance of different domains of the RAD54 protein, a member of a large protein family including the protein mutated in Cockayne's syndrome B, the interactions between proteins involved in recombinational repair, and the order of function of genes in this pathway. The proposed experiments will give us additional information about this important repair pathway. The RAD54 gene is necessary for recombinational repair, but it is not widely studied. Molecular and biochemical studies will be initiated to examine interesting regions encoding a putative nucleotide-binding domain, potential DNA helicase domains, and a probable zinc finger. Site specific mutagenesis using PCR primers will be used to introduce mutations into potential domains. These mutations will be tested for their effect on repair. Low fidelity PCR will be used to isolate random rad54 mutations, which will be screened for temperature-conditional and leaky alleles. Interesting alleles will be sequenced and used in other experiments. In conjunction with these molecular experiments, we will also isolate the RAD54 protein and a truncated form containing the zinc finger region. We will attempt to isolate RAD54 from E. coli, but use overexpression in yeast if necessary. This protein will be characterized for several activities, including ATPase activity, zinc binding, binding to strand breaks, and helicase activity. Some mutant RAD54 proteins will also be characterized. The RAD52 and RAD51 proteins have recently been reported to interact. -We plan to determine if this protein interaction is essential for recombinational repair, by further characterizing our rad52-20 allele which may be defective in this interaction. To test if the rad52-20 protein has weakened interaction with RAD51, we will use the molecular two-hybrid system. Studies will also be initiated to determine if the RAD54 protein interacts with other proteins, using both genetic reversion of conditional rad54 alleles and the two-hybrid system. Experiments are proposed to examine the functional order of the RAD51, 52 and 54 genes, using temperature shift experiments in conjunction with temperature-sensitive (t.s.) and cold-sensitive (c.s.) alleles. We plan to determine if RAD54 functions before or after RAD51 and RAD52, using our recently isolated c.s. and t.s. rad51 alleles in combination with the t.s. rad54-3 allele and new t.s. rad52 alleles, isolated by Kaytor and Livingston.