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

重组修复，包括姐妹染色单体交换，是 酵母中的三个主要修复途径，对修复是至关重要的 X射线诱导的双链断裂，并参与修复 包括DNA交联链在内的多种化学损伤。 重组修复可能也被用于哺乳动物细胞，并且 最近，人类基因被分离出来，它们是这三个基因的同源物。 我们将研究酵母基因。因为几个修复基因已经被 与癌症的发生密切相关，更好地理解了 修复基因的功能应该会给我们提供重要的信息 对癌症的发生和细胞如何处理都有影响 暴露在环境致癌物质中。关于重组的研究 酿酒酵母的修复，强调三个基因(RAD51， 52和54)是这一进程最必要的，将继续进行。这个 具体目标涉及RAD54不同领域的重要性 蛋白质，包括突变的蛋白质在内的一个大蛋白质家族的成员 在Cockayne‘s综合征B中，参与的蛋白质之间的相互作用 重组修复，以及基因在其中的功能顺序 路径。拟议的实验将给我们提供更多的信息 关于这条重要的修复途径。 RAD54基因是重组修复所必需的，但它不是 被广泛研究。将启动分子和生化研究，以 研究编码假定的核苷酸结合的有趣区域 潜在的DNA解旋酶结构域，以及可能的锌指。立地 使用聚合酶链式反应的特异性诱变将被用来介绍 突变为潜在的结构域。将对这些突变进行检测 它们对修复的影响。将使用低保真聚合酶链式反应分离随机 Rad54突变，将对温度条件和 泄露的等位基因。有趣的等位基因将被测序并用于其他 实验。结合这些分子实验，我们将 还分离了RAD54蛋白和含有锌的截短形式 手指区域。我们将尝试从大肠杆菌中分离RAD54，但使用 如有必要，在酵母中过度表达。这种蛋白质的特征将是 对于几种活性，包括ATPase活性、锌结合、结合 链断裂和解旋酶活性。一些突变的RAD54蛋白将 也是具有特征的。 RAD52和RAD51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 重组修复，通过进一步鉴定我们的rad52-20等位基因 其在这种相互作用中可能是有缺陷的。以测试RAD52-20是否 蛋白质已经减弱了与RAD51的相互作用，我们将使用分子 双杂交系统。还将启动研究，以确定是否 RAD54蛋白与其他蛋白相互作用，利用两种遗传逆转 有条件的rad54等位基因和双杂交系统。 建议进行实验以检查RAD51的功能顺序， 52和54个基因，使用温度变化实验结合 温敏性(T.S.)和冷敏感(C.S.)等位基因。我们计划 要确定RAD54在RAD51和RAD52之前或之后是否起作用，请使用 我们最近被隔离的C.S.和T.S.RAD51等位基因与 T.S.RAD54-3等位基因和新T.S.RAD52等位基因，由Kaytor和 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.