MOLECULAR MECHANISMS OF DNA REPAIR AND RECOMBINATION IN YEAST

我们正在调查与此相关的独特的染色体代谢事件 与减数分裂和接触DNA后染色体的修复有关 破坏性因素。修复DNA双链所需的许多基因 链断裂(DSB)是成功完成 减数分裂周期。已经鉴定出两种蛋白质/酶，它们似乎可以 在减数分裂和/或修复中起作用；依赖镁(2)的核酸酶(RhoNUC， 以前被称为yNucR，以及一种能够执行 链交换反应(SEP)。这两种蛋白质的活性都出现了 在RAD52基因的控制下，这是一种修复 辐射诱导的双链断裂和减数分裂的完成。为了调查 RAD52的作用，我们创造了含有染色体驻留的菌株 RAD52和RAD52基因在载体上的缺失 酵母胆汁促进剂。这使我们能够严格地调节表达 RAD52基因。我们发现，对于DSB的修复，存在 在电离辐射治疗之前，需要RAD52蛋白的含量。 有趣的是，RhoNUC蛋白的表达不需要 功能性RAD52基因。这表明RAD52对RhoNUCH的控制是 不是在转录或翻译层面上。为了辨认 其他可能与RAD52蛋白相互作用的蛋白质，我们已经开始 筛选“蛋白质相互作用”文库。这个系统将允许我们 确定与RAD52物理上相互作用的其他蛋白质。 RAD52在DNA复制过程中的可能作用也是 调查过了。使用一种独特的系统来测量频率 将细菌转座子TN5“切除”到酵母LYS2基因， 我们已经发现了编码DNA聚合酶I或III的基因突变 导致高水平的TN5“切除”。没有观察到DNA的增加 聚合酶II突变体。在POLIII中看到的高水平的“切除” 当RAD52的零突变体被引入时，Poli菌株减少 这些菌株。这表明了所提出的酶之间的相互作用 负责DNA滞后链合成(POLIII和POLI)和 RAD52。目前，我们正在调查这种相互作用的性质。

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. Two proteins/enzyme have been identified that appear to play a role in meiosis and/or repair; a Mg(2+) dependent nuclease (RhoNUC, previously referred to as yNucR, and a protein that is able to carry out a strand exchange reaction (SEP). Both of these protein activities appears to under the control of the RAD52 gene, a gene required for the repair of radiation-induced DSBs and the completion of meiosis. To investigate the role of RAD52, we have created strains containing a chromosomal resident deletion of the RAD52 and a RAD52 gene on a plasmid under the control of the yeast GALl 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 expression of the RhoNUC protein does not require a functional RAD52 gene. This suggests the control of RhoNUCH by RAD52 is not at the transcriptional or translational level. In order to identify other proteins which may interact with the RAD52 protein, we have begun screening a "protein interaction" library. This system will allow us to identify other proteins which physically interact with RAD52. The possible role of RAD52 during DNA 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 the mutations in the genes 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 polIII or polI 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 (POLIII and POLI) and RAD52. Currently we are investigating the nature of this interaction.