Recruitment of End-Processing Factors in DSB Repair

DSB 修复中末端加工因子的招募

基本信息

批准号：
8919907
负责人：
Paula Louise Fischhaber
金额：
$ 10.2万
依托单位：
CALIFORNIA STATE UNIVERSITY NORTHRIDGE
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-01 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8919907
关键词：
Address Affect Aging Area Arginine Ataxia Telangiectasia Binding Biochemical Biochemistry Biological Cancer Etiology Capital Cell Cycle Cells Chemical Agents Chromosome abnormality Chromosomes Clinical Complex Critical Pathways DNA DNA Damage DNA Repair DNA Sequence DNA annealing DNA lesion DNA strand break Data Defect Double Strand Break Repair Drug Targeting Elderly Excision Family Fanconi&apos s Anemia Fluorescence Microscopy Genes Genetic Genetic Materials Genome Glycine Homologous Gene Human In Vitro Investigation Ionizing radiation Label Laboratories Length Letters Life Location Malignant Neoplasms Methods Modeling Molecular Monitor Mutagenesis Mutation Names Nomenclature Pathogenesis Pathway interactions Pattern Pharmacologic Substance Phase Predisposition Prevention strategy Process Proteins Rad52 protein Recruitment Activity Regulation Reporting Research Role Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Severities Signal Transduction Single-Stranded DNA Site Source Surgical Flaps Symptoms Tail Techniques Work Yeast Model System Yeasts base cancer prevention cancer risk cancer therapy endodeoxyribonuclease SceI endonuclease fluorescence imaging genetic information in vivo innovation mutant prevent protein complex protein function protein protein interaction public health relevance repaired research study restoration restriction enzyme

项目摘要

DESCRIPTION (provided by applicant): Ionizing radiation and chemical agents induce strand breaks in DNA, which give rise to mutations and chromosomal alterations that can cause cancer. One of the chief biological defenses against DNA strand breaks is Double-Strand Break (DSB) repair, a complicated family of biologic pathways that repairs DNA strand breaks. Some modes of DSB Repair can proceed with full restoration of the DNA sequence and no resulting loss of genetic information, but others result in significant loss. The molecular underpinnings of DSB repair are a matter of intense study, but questions remain regarding biochemical requirements and pathway selection, given that they are not all equally effective in repairing chromosomes without loss of genetic information. Within these broader issues are more specific questions regarding the mechanism of recruitment of proteins that participate in some DSB repair pathways but not others. In baker's yeast (S. cerevisiae), Saw1 protein recruits the Rad1-Rad10 protein complex to DSB sites, probably by binding to Rad52 protein, but the biochemical details regarding how this occurs are not known in detail. This project will detail the molecular basis for recruitment of the Saw1, Rad10 and Rad52 proteins to sites of DSB repair in the yeast S. cerevisiae. The specific aims of this proposal are to determine in an evolutionarily-conserved eukaryotic model system, the yeast, S. cerevisiae: 1) whether patterns of Saw1 and Rad10 recruitment to DSBs are altered depending on the length of the nonhomologous DNA sequence flanking the DSB site, 2) minimum binding domains of Rad52 and Saw1 required to form a Rad52-Saw1 complex, 3) whether human Rad52 can be recruited to yeast DSB sites in the absence of yeast Rad52 and 4) whether human Rad52 can recruit Saw1 to yeast DSB sites, and whether mutations that result in loss of the "strand annealing" function of Rad52 affect recruitment of Saw1. These aims will be investigated primarily by innovative fluorescence microscopy experiments in which DSBs will be induced specifically at fluorescently labeled loci on different chromosomes in live yeast cells, and their repair monitored by fluorescence imaging of convergent fluorescent signals. Localization of fluorescently labeled Rad52, Saw1 and Rad10 to the DSBs will be used to study the effects on recruitment of varying the lengths of nonhomologous DNA sequences flanking the DSBs as well as mutations in the RAD52 and SAW1 genes. In vitro techniques will be used to further study important interactions between the Rad52 and Saw1 proteins. These experiments will address important questions regarding the biochemical requirements for recruitment of Rad52, Saw1 and Rad10 to DSB sites. Understanding the molecular basis for cancer and aging is important for advancing clinical strategies to minimize human suffering. A more detailed understanding of the mechanisms by which cells repair DNA will aid in finding new drug targets for pharmaceuticals that might minimize cancer risk and the ailments associated with aging.

描述（由申请人提供）：电离辐射和化学试剂会引起 DNA 链断裂，从而引起突变和染色体改变，从而导致癌症。针对 DNA 链断裂的主要生物防御之一是双链断裂 (DSB) 修复，这是修复 DNA 链断裂的一类复杂的生物途径。 DSB 修复的某些模式可以完全恢复 DNA 序列，并且不会导致遗传信息丢失，但其他模式会导致重大损失。 DSB 修复的分子基础是一个需要深入研究的问题，但关于生化要求和途径选择的问题仍然存在，因为它们在修复染色体而不丢失遗传信息方面并非都同样有效。在这些更广泛的问题中，有一些更具体的问题，涉及参与某些 DSB 修复途径但不参与其他途径的蛋白质的招募机制。在面包酵母（酿酒酵母）中，Saw1 蛋白可能通过与 Rad52 蛋白结合，将 Rad1-Rad10 蛋白复合物募集到 DSB 位点，但有关此过程如何发生的生化细节尚不清楚。该项目将详细介绍将 Saw1、Rad10 和 Rad52 蛋白招募到酿酒酵母 DSB 修复位点的分子基础。该提案的具体目的是确定在进化保守的真核模型系统中，酵母，酿酒酵母：1) Saw1 和 Rad10 招募到 DSB 的模式是否会根据 DSB 侧翼的非同源 DNA 序列的长度而改变位点，2) 形成 Rad52-Saw1 复合物所需的 Rad52 和 Saw1 的最小结合域，3) 人 Rad52 是否可以在没有酵母 Rad52 的情况下招募到酵母 DSB 位点，4) 人类 Rad52 是否可以将 Saw1 招募到酵母 DSB 位点，以及导致 Rad52 丧失“链退火”功能的突变是否会影响 Saw1 的招募。这些目标将主要通过创新的荧光显微镜实验进行研究，其中 DSB 将在活酵母细胞中不同染色体上的荧光标记位点处被特异性诱导，并通过会聚荧光信号的荧光成像监测它们的修复。荧光标记的 Rad52、Saw1 和 Rad10 在 DSB 上的定位将用于研究 DSB 侧翼不同长度的非同源 DNA 序列的招募以及 RAD52 和 SAW1 基因突变的影响。体外技术将用于进一步研究 Rad52 和 Saw1 蛋白之间的重要相互作用。这些实验将解决有关将 Rad52、Saw1 和 Rad10 招募到 DSB 位点的生化要求的重要问题。了解癌症和衰老的分子基础对于推进临床策略以尽量减少人类痛苦非常重要。更详细地了解细胞修复 DNA 的机制将有助于寻找新的药物靶标，从而最大限度地降低癌症风险和与衰老相关的疾病。