DOUBLE STRAND BREAKS AND HETERODUPLEX DNA IN YEAST

酵母中的双链断裂和异源双链 DNA

基本信息

批准号：
6386822
负责人：
ANDREW VERSHON
金额：
$ 14.65万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至 2003-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6386822
关键词：
DNA binding protein DNA damage DNA repair SDS polyacrylamide gel electrophoresis Saccharomyces cerevisiae electron microscopy fungal genetics gel mobility shift assay genetic mapping genetic recombination molecular cloning nucleic acid structure protein sequence protein structure function southern blotting western blottings

项目摘要

Exposure of cells to ionizing radiation (X-rays and gamma-rays) results in the formation of double-stranded breaks (DSBs) in chromosomal DNA, a particularly deleterious form of genomic injury. In all organisms, one of the main ways to repair DSBs is by homologous recombination using undamaged DNA present elsewhere in the cell. The purpose of the experiments in this proposal is to analyze the enzymology of this reaction in the yeast Saccharomyces cerevisiae. Specific Aim I: One of our goals is to identify and understand proteins that assemble at DSBs and catalyze early steps in their repair. To address this issue, we have been utilizing a powerful model system for recombinational repair: mating-type switching. We have identified a new protein, called YZ binding protein, which is required for DSB formation in this system. In the first Specific Aim, we describe experiments to analyze the function of this protein in detail. These studies will allow us to progress to the identification and characterization of additional factors involved in early steps of DSB repair. Specific Aim II: Another of our goals is to understand proteins that generate heteroduplex DNA (hDNA), the central intermediate in recombinational repair. Using an in vitro D-loop assay, we have purified a large complex from yeast extracts that catalyzes hDNA formation in an ATP- and homology-dependent reaction. Optimal product formation by this complex requires functional Rad51p and Rad52p, each of which is known to play an essential role in recombinational repair. In the second Specific Aim, we describe experiments to further characterize this activity. Analysis of such a "recombination machine" will dramatically improve our understanding of the enzymology of recombinational repair. Significance: DSBs are extremely dangerous to the cell. They can, for instance, induce cell death or genomic rearrangements if left unrepaired. DSBs also have positive roles in cells, i.e., they initiate the meiotic recombination reaction required for the proper segregation of chromosomes into gametes. Finally, it has been demonstrated that human Rad51p, a central protein in recombinational repair, is associated in vivo with the BRCA1 and BRCA2 genes, suggesting that breast cancer results from a defect in recombinational repair. The analysis of recombinational-repair proteins in simpler and more tractable organisms, like yeast, will therefore provide invaluable insights into several fundamental and important cellular processes.

细胞暴露于电离辐射（X射线和伽马射线）结果在染色体DNA中双链断裂（DSB）的形成中，一种特别有害的基因组损伤形式。在所有生物中，修复DSB的主要方法之一是使用同源重组细胞中其他地方的未损坏的DNA。目的该提案的实验是分析此的酶学酿酒酵母的酵母糖粉中的反应。特定目标I：之一我们的目标是识别和理解在DSB上组装的蛋白质并催化了他们的修复阶段。为了解决这个问题，我们一直在利用强大的模型系统进行重组维修：交配型切换。我们已经确定了一种称为Yz的新蛋白结合蛋白，这是该系统中DSB形成所必需的。在第一个特定目的中，我们描述了分析的实验该蛋白质的功能详细。这些研究将使我们能够进展到附加的识别和表征 DSB修复的早期步骤涉及的因素。特定目标II：另一个我们的目标是了解产生异质双链DNA的蛋白质（HDNA），重组修复中的中间中间。使用一个在体外D-loop测定法，我们从酵母中纯化了一个大复合物催化HDNA在ATP和同源性依赖性的提取物反应。该复合物的最佳产品形成需要功能 RAD51P和RAD52P，每一个都在重组维修。在第二个特定目标中，我们描述实验进一步表征了这一活动。这种分析 “重组机器”将极大地提高我们对重组修复的酶学。意义：DSB对细胞非常危险。他们可以实例，如果离开，诱导细胞死亡或基因组重排未修复。 DSB在细胞中也具有积极的作用，即它们启动适当分离所需的减数分裂重组反应染色体成配子。最后，已经证明人Rad51p是一种重组维修的中心蛋白，与带有BRCA1和BRCA2基因的体内，表明乳腺癌重组修复缺陷的结果。分析重组修复蛋白中的更简单，更可拖动的生物，因此，像酵母一样，将为几种提供宝贵的见解基本和重要的细胞过程。