Recruitment of Rad10 in Double-strand Break Repair

描述（由申请人提供）：电离辐射和化学试剂诱导DNA链断裂，从而引起突变和染色体改变，从而导致癌症。DNA链断裂的主要生物防御之一是双链断裂修复（DSBR），这是一个复杂的修复DNA链断裂的生物途径家族。一些模式的DSBR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着的染色体丢失。DNA修复领域的研究人员正在阐明DSBR的分子基础，但许多问题仍然存在，包括生化要求、途径的时机，甚至如何选择最合适的途径，因为它们在不丢失遗传信息的情况下修复染色体时并非都同样有效。在这个问题的保护伞下，更具体的问题是关于参与某些途径而不参与其他途径的蛋白质的定时和招募机制。本提案的具体目的是确定在进化保守的真核生物模型系统中，酵母，酿酒酵母：1)是否Rad10招聘模式的双链断裂(双边带)改变依赖的程度和邻近DNA序列同源性在争端解决机构网站,侧面2)是否Rad10招聘“长串退火”(SSA)的双边带修复模式是依赖于SAW1基因,以及是否Rad10和SAW1蛋白质colocalize SSA维修地点和3)Rad10招聘双边带网站是否依赖于基因SLX4 Rad10是否和SLX4蛋白质colocalize双边带网站。这些目标将主要使用创新的荧光显微镜实验进行研究，在该实验中，链断裂将被诱导，特别是在活酵母细胞中，DNA修复位置将被活细胞的荧光成像监测。荧光标记蛋白的行为将在含有野生型和突变基因的适当组合以及改变dsb两侧的DNA序列的菌株中进行比较。荧光标记或突变基因的功能特征将通过基于质粒的DNA损伤诱导/修复试验进行研究，该试验将通过Southern杂交进行分析。这些实验将解决在DSBR的几个关键模式/背景下与Rad10的时间和招募有关的生化需求的重要问题，并描述Rad10如何被招募到DSB位点的差异。癌症和衰老的分子基础是一个重要的研究领域，以推进临床策略，将人类的痛苦降到最低。更详细地了解细胞修复DNA的生物化学过程，将有助于找到新的药物靶点，从而最大限度地降低癌症风险和与衰老有关的疾病。

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.