Recruitment of End-Processing Factors in DSB Repair

DSB 修复中末端加工因子的招募

• 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
• 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' 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

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.

说明（由申请方提供）：电离辐射和化学制剂会诱导DNA链断裂，从而导致突变和染色体改变，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链断裂的复杂生物途径家族。一些DSB修复模式可以进行DNA序列的完全恢复，而不会导致遗传信息的丢失，但其他模式会导致显著的丢失。DSB修复的分子基础是一个深入研究的问题，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不都同样有效。 在这些更广泛的问题中，有一些更具体的问题是关于参与一些DSB修复途径而不是其他途径的蛋白质的募集机制。在面包酵母（S. Saw 1蛋白将Rad 1-Rad 10蛋白复合物募集到DSB位点，可能是通过与Rad 52蛋白结合，但关于这是如何发生的生物化学细节尚不清楚。该项目将详细介绍 Saw 1、Rad 10和Rad 52蛋白在酵母S.酿酒的。该建议的具体目标是确定在进化保守的真核模型系统中，酵母，S。酿酒酵母：1）Saw 1和Rad 10募集至DSB的模式是否根据DSB位点侧翼的非同源DNA序列的长度而改变，2）形成Rad 52-Saw 1复合物所需的Rad 52和Saw 1的最小结合结构域，3）在不存在酵母Rad 52的情况下，人Rad 52是否可以募集到酵母DSB位点，和4）人Rad 52是否可以募集Saw 1到酵母DSB位点，以及导致Rad 52“链退火”功能丧失的突变是否影响Saw 1的募集。 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特异性地诱导，并通过会聚荧光信号的荧光成像监测其修复。将荧光标记的Rad 52、Saw 1和Rad 10定位于DSB，以研究不同长度的DSB侧翼非同源DNA序列以及RAD 52和SAW 1基因突变对招募的影响。体外技术将用于进一步研究Rad 52和Saw 1蛋白之间的重要相互作用。这些实验将解决有关招募Rad 52，Saw 1和Rad 10的DSB网站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最大限度地减少人类痛苦非常重要。更详细地了解细胞修复DNA的机制将有助于为药物寻找新的药物靶点，从而最大限度地降低癌症风险和与衰老相关的疾病。