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

酵母 DNA 修复基因 RAD51、52 ​​和 54 的分析

• 批准号: 2021916
• 负责人: DAVID SCHILD
• 金额: $ 27.81万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-03-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2021916
• 关键词: DNA binding protein; DNA damage; DNA repair; Escherichia coli; Saccharomyces; X ray; adenosinetriphosphatase; alleles; enzyme activity; fungal genetics; gene mutation; genetic recombination; genetic strain; helicase; intermolecular interaction; molecular site; nucleic acid sequence; polymerase chain reaction; protein structure function; radiation genetics; radiation sensitivity; site directed mutagenesis; temperature sensitive mutant

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.

重组维修，包括姐妹 - 染色质交换，是 酵母中的三个主要维修途径，对于修复至关重要 X射线诱导的双链断裂，还参与了修复 在许多类型的化学损伤中，包括DNA交联。 重组维修可能也可能用于哺乳动物细胞，并且 最近已经孤立了人类基因，这是三个的同源物 我们将研究的酵母基因。由于几个修复基因已经 对癌变有着强烈的影响，更好地理解了 维修基因功能应为我们提供重要信息 对癌变和细胞如何处理都有影响 暴露于环境致癌物。重组研究 酿酒酵母的酵母菌修复，强调三个基因（rad51， 52和54）将继续进行此过程。这 具体目的与Rad54不同域的重要性有关 蛋白质，包括蛋白质突变的大蛋白质家族的成员 在Cockayne的综合征B中，涉及的蛋白质之间的相互作用 重组修复和基因的功能顺序 路径。拟议的实验将为我们提供其他信息 关于这个重要的维修途径。 Rad54基因是重组维修所必需的，但不是 广泛研究。分子和生化研究将开始 检查编码假定核苷酸结合的有趣区域 结构域，潜在的DNA解旋酶结构域和可能的锌指。地点 使用PCR引物的特定诱变将用于引入 突变进入潜在域。这些突变将进行测试 它们对维修的影响。低保真度PCR将用于隔离随机 RAD54突变，将筛选以获取温度条件和 泄漏的等位基因。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 还隔离Rad54蛋白和包含锌的截短形式 手指区域。我们将尝试将Rad54与大肠杆菌分离，但使用 如有必要，酵母中的过表达。该蛋白将被表征 对于几种活动，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 也被描述。 最近据报道Rad52和Rad51蛋白相互作用。 -我们 计划确定这种蛋白质相互作用是否对 重组维修，通过进一步表征我们的Rad52-20等位基因 在这种相互作用中可能有缺陷。测试Rad52-20是否 蛋白质与RAD51的相互作用削弱了，我们将使用分子 两个杂交系统。还将开始研究 Rad54蛋白使用两种遗传回归与其他蛋白质相互作用 有条件的RAD54等位基因和两个杂交系统。 提出了实验来检查RAD51的功能顺序 52和54基因，使用温度转移实验与 温度敏感（T.S.）和冷敏感（C.S.）等位基因。我们计划 为了确定RAD54在Rad51和Rad52之前或之后是否功能 我们最近孤立的C.S.和T.S. RAD51等位基因与 T.S. RAD54-3等位基因和新的T.S. RAD52等位基因，由Kaytor隔离和 利文斯顿。