Recruitment of Rad10 in Double-strand Break Repair

双链断裂修复中 Rad10 的招募

基本信息

批准号：
8508329
负责人：
Paula Louise Fischhaber
金额：
$ 0.41万
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 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8508329
关键词：
Address Aging Appearance Area Ataxia Telangiectasia Automobile Driving Behavior Biochemical Biochemistry Biological Biological Assay CCL7 gene California Cancer Etiology Capital Cell Cycle Cells Chemical Agents Chromatin Chromosomal Loss Chromosome abnormality Chromosomes Clinical Complex DNA DNA Damage DNA Double Strand Break DNA Repair DNA Sequence DNA lesion DNA strand break Data Development Double Strand Break Repair Drug Delivery Systems Elderly Eukaryota Event Family Fanconi&apos s Anemia Fluorescence Microscopy Foundations Funding Genes Genetic Genetic Materials Genome Goals Grant Homologous Gene Human Investigation Ionizing radiation Knowledge Label Laboratories Lead Letters Life Location Malignant Neoplasms Manuscripts Mentors Mentorship Methodology Methods Minority Modeling Molecular Monitor Mutagenesis Mutation Names Nucleotide Excision Repair Pathogenesis Pathway interactions Pattern Peer Review Pharmacologic Substance Phase Phosphorylation Plasmids Postdoctoral Fellow Predisposition Prevention strategy Proteins Publications Publishing Recruitment Activity Regulation Research Research Personnel Role Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Sequence Homology Severities Signal Transduction Site Source Stream Students Symptoms Talents Text Time Universities Work Yeast Model System Yeasts authority base cancer prevention cancer risk cancer therapy endodeoxyribonuclease SceI endonuclease fluorescence imaging homologous recombination in vivo innovation mutant novel prevent professor programs protein function public health relevance repaired research study restoration southern hybridization

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 Repair (DSBR), a complicated family of biologic pathways that repairs DNA strand breaks. Some modes of DSBR can proceed with full restoration of the DNA sequence and no resulting loss of genetic information, but others result in significant chromosomal loss. The molecular underpinnings of DSBR are being elucidated by researchers in the DNA Repair field, but many questions remain regarding biochemical requirements, timing of the pathways, and even how the most appropriate pathway is selected, given that they are not all equally effective in repairing chromosomes without loss of genetic information. Under this umbrella of interrogation are more specific questions regarding the mechanism for timing and recruitment of proteins that participate in some pathways but not others. The specific aims of this proposal are to determine in an evolutionarily-conserved eukaryotic model system, the yeast, S. cerevisiae: 1) whether patterns of Rad10 recruitment to Double-Strand Breaks (DSBs) are altered depending on the extent and proximity of DNA sequence homology flanking the DSB site, 2) whether Rad10 recruitment in the "single-strand annealing" (SSA) mode of DSB repair is dependent on the SAW1 gene, and whether Rad10 and Saw1 proteins colocalize at SSA repair sites and 3) whether Rad10 recruitment to DSB sites is dependent on the gene SLX4 and whether Rad10 and Slx4 proteins colocalize at DSB sites. These aims will be investigated primarily using innovative fluorescence microscopy experiments in which strand breaks will be induced site-specifically in live yeast cells and DNA repair locations will be monitored by fluorescence imaging of the live cells. The behavior of the fluorescently labeled proteins will be compared in strains containing the appropriate combinations of wild-type and mutant genes and varying the DNA sequences flanking the DSBs. Characterization of the functionality of fluorescently-labeled or mutant genes will be investigated with a plasmid-based DNA damage induction/ repair assay which will be analyzed by Southern hybridization. These experiments will address important questions regarding the biochemical requirements bearing on the timing and recruitment of Rad10 in several key modes/contexts of DSBR and delineate the differences in how Rad10 is recruited to DSB sites. The molecular basis for cancer and aging is an important area of research in order to advance clinical strategies to will minimize human suffering. A more detailed understanding of the biochemistry 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. PUBLIC HEALTH RELEVANCE: The proposed research will increase our understanding of the mechanism of regulation of a complex biological pathway that protects against cancer and aging known as Double-strand Break Repair. Results of this work may aid in developing cancer prevention strategies and methods to reduce suffering in the elderly by preventing some of the symptoms of 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能力之一是双链断裂修复（DSBR），这是一个复杂的生物学途径家族，可修复DNA链断裂。 DSBR的某些模式可以进行完全恢复DNA序列，而不会导致遗传信息的丢失，而另一些则导致显着的染色体损失。 DSBR的研究人员阐明了DSBR的分子基础，但是鉴于它们在维修染色体方面并非同样有效而不会损失遗传信息。在这种审讯的保护下，关于参与某些途径而不是其他途径的蛋白质定时和募集机制的更具体问题。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，酿酒酵母：1）RAD10募集到双链断裂（DSB）的模式是否会根据DNA序列的范围和接近性，是否会改变DNA序列的程度和接近性。 DSB修复取决于SAW1基因，RAD10和SAW1蛋白是否在SSA修复位点共定位以及3）RAD10募集到DSB的募集是否取决于基因SLX4以及RAD10和SLX4蛋白是否在DSB位点共定位。这些目标将主要使用创新的荧光显微镜实验进行研究，其中将在活酵母细胞中特定于现场诱导链断裂，而DNA修复位置将通过活细胞的荧光成像来监测。将在包含野生型和突变基因的适当组合的菌株中比较荧光标记的蛋白质的行为，并改变DSB侧面的DNA序列。荧光标记或突变基因功能的表征将通过基于质粒的DNA损伤诱导/修复测定法进行研究，该测定法将通过南部杂交分析。这些实验将解决有关在DSBR的几种关键模式/上下文中与RAD10的时间和募集有关的生化要求的重要问题，并描绘了RAD10招募到DSB站点的差异。癌症和衰老的分子基础是研究的重要领域，以促进临床策略以最大程度地减少人类痛苦。对生物化学的更详细的了解，细胞修复DNA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靶标。公共卫生相关性：拟议的研究将提高我们对一种复杂生物学途径调节机制的理解，该途径可以防止癌症和称为双链断裂修复的癌症和衰老。这项工作的结果可能有助于制定预防癌症的策略和方法，以防止某些衰老症状减少老年人的痛苦。