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在DNA中诱导链断裂，这会导致可能导致癌症的突变和染色体改变。针对DNA链断裂的主要生物防御措施之一是双链断裂（DSB）修复，这是一个复杂的生物学途径家族，可修复DNA链断裂。 DSB修复的某些模式可以进行DNA序列的完全恢复，而不会导致遗传信息的丢失，但其他模式则导致了重大损失。 DSB修复的分子基础是一项激烈的研究问题，但是关于生化需求和途径选择的问题仍然存在，因为它们并非在修复染色体而不会丢失遗传信息方面同样有效。在这些更广泛的问题中，关于参与某些DSB修复途径的蛋白质机制的更具体问题，但不是其他问题。在贝克的酵母（酿酒酵母）中，SAW1蛋白将Rad1-Rad10蛋白复合物募集到DSB位点，可能是通过与RAD52蛋白结合的，但是有关这种发生方式的生化细节尚不清楚。该项目将详细介绍将SAW1，RAD10和RAD52蛋白募集到酵母菌酿酒酵母中DSB修复位点的分子基础。该提案的具体目的是在进化保存的真核生物模型系统中确定酵母，S。cerevisiae：1）SAW1和RAD10募集到DSB的模式是否是否会根据非原理DNA序列的长度而改变DSB的长度。可以在没有酵母Rad52和4）的情况下招募到酵母DSB位点，而人Rad52是否可以募集SAW1到酵母DSB位点，以及是否导致RAD52的“链退火”功能的突变是否会影响SAW1的募集。这些目标将主要通过创新的荧光显微镜实验研究，其中DSB将在活酵母细胞中的不同染色体上专门在荧光标记的基因座上诱导，并通过收敛荧光信号的荧光成像监测其修复。将荧光标记为RAD52，SAW1和RAD10的荧光定位将用于研究对DSB和RAD52和SAW1基因突变的非同源DNA序列的募集的影响。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DSB站点募集RAD52，SAW1和RAD10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程度地减少人类痛苦至关重要。对细胞修复DNA修复DNA的机制的更详细的理解将有助于寻找可能最大程度地减少癌症风险和与衰老相关的疾病的药物的新药物。