MOLECULAR MECHANISMS OF DNA REPAIR AND RECOMBINATION IN YEAST

酵母 DNA 修复和重组的分子机制

• 批准号: 3841142
• 负责人: M A RESNICK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3841142
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; DNA replication; chromosome aberrations; fungal genetics; gene deletion mutation; gene expression; gene induction /repression; genetic recombination; meiosis; nuclease

We are investigating the unique chromosomal metabolic events associated with meiosis and the repair of chromosomes following exposure to DNA damaging agents. Many of the genes necessary for the repair of DNA double strand breaks (DSB) are required for the successful completion of the meiotic cycle. In particular the RAD52 gene is essential for the repair of DSBs and the successful completion of meiosis. Furthermore, RAD52 appears to influence mitotic recombination. To investigate the role of a RAD52, we have created strains containing a chromosomal resident deletion of RAD52 and a RAD52 gene under the control of the yeast GAL1 promoter. This allows us to tightly regulate the expression of the RAD52 gene. We have found that for the repair of DSBs the presence of RAD52 protein is required prior to treatment with ionizing radiation. Interestingly, the presence of a previously isolated nuclease (RhoNUC) appears to be influenced by RAD52, however, this control appears to be post-translational. In order to identify other proteins which may interact with RAD52 we have begun screening a "protein interaction" library. The possible role of RAD52 during replication is also being investigated. Using a unique system whereby we measure the frequency of "excision" of a bacterial transposon Tn5 placed into the yeast LYS2 gene, we have found that mutations in the gene encoding DNA polymerase I or III lead to high levels of Tn5 "excision". No increase is observed in DNA polymerase II mutants. The elevated levels of "excision" seen in po1III or po1I strains is reduced when a null mutant of RAD52 is introduced into these strains. This suggests an interaction between the enzymes proposed to be responsible for DNA lagging strand synthesis and RAD52.

我们正在研究与此相关的独特的染色体代谢事件 与减数分裂和染色体修复接触DNA后 破坏剂。 许多修复DNA所必需的基因 链断裂（DSB）是成功完成 减数分裂周期 特别是RAD52基因是修复 DSB和减数分裂的成功完成。 此外，RAD52似乎 影响有丝分裂重组。 为了研究RAD 52的作用，我们 已经创建了含有RAD52染色体常驻缺失的菌株 和在酵母GAL 1启动子控制下的RAD 52基因。 这允许 我们严格调控RAD52基因的表达。 我们发现 对于DSB的修复，在修复之前需要RAD52蛋白的存在。 电离辐射治疗 有意思的是， 先前分离的核酸酶（RhoNUC）似乎受到RAD 52的影响， 然而，这种控制似乎是翻译后的。 为了 鉴定其他可能与RAD52相互作用的蛋白质， 筛选“蛋白质相互作用”文库。 RAD52在复制过程中的可能作用也正在研究中。 使用一个独特的系统，我们测量的频率“切除”的 细菌转座子Tn5放入酵母LYS2基因，我们发现 编码DNA聚合酶I或III的基因突变导致高水平的 Tn5“切除”水平。 未观察到DNA聚合酶II增加 变种人 在po1III或po1I菌株中观察到的“切除”水平升高 当将RAD 52的无效突变体引入这些菌株中时， 这表明，酶之间的相互作用被认为是负责 用于DNA滞后链合成和RAD52。