Recruitment of Rad10 in Double-strand Break Repair

描述(由申请人提供)：电离辐射和化学物质会导致DNA链断裂，从而引起突变和染色体变化，从而导致癌症。针对DNA链断裂的主要生物防御措施之一是双链断裂修复(DSBR)，这是一个复杂的生物通路家族，用于修复DNA链断裂。DSBR的一些模式可以完全恢复DNA序列，不会导致遗传信息的丢失，但另一些模式会导致显著的染色体丢失。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，如生化要求、途径的时机，甚至如何选择最合适的途径，因为它们在修复染色体而不丢失遗传信息方面并不都是同样有效的。在这个审问的保护伞下，是关于参与某些途径而不是另一些途径的蛋白质的定时和招募机制的更具体的问题。本建议的具体目的是在一个进化保守的真核模型系统中确定酿酒酵母：1)Rad10招募到双链断裂(DSB)的模式是否根据DSB位点两侧DNA序列同源的程度和邻近程度而改变，2)DSB修复的“单链退火”(SSA)模式中的Rad10招募是否依赖于SAW1基因，Rad10和Saw1蛋白是否共存于SSA修复位点，3)Rad10募集到DSB位点是否依赖于基因SLX4，以及Rad10和Slx4是否共存于DSB位点。这些目标将主要通过创新的荧光显微镜实验来研究，在这些实验中，链断裂将被特定地诱导在活酵母细胞中，DNA修复位置将通过活细胞的荧光成像进行监测。荧光标记蛋白质的行为将在含有适当的野生型和突变基因组合的菌株中进行比较，并改变DSB两侧的DNA序列。荧光标记或突变基因的功能特性将通过基于质粒的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。这些实验将解决有关在DSBR的几种关键模式/背景下对Rad10的时间和招募产生影响的生化要求的重要问题，并描绘如何将Rad10招募到DSB站点的差异。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，将人类的痛苦降至最低。对细胞修复DNA的生物化学有更详细的了解，将有助于为药物寻找新的药物靶点，可能会将癌症风险和与衰老相关的疾病降至最低。 与公共健康相关：拟议中的研究将增加我们对一种复杂生物途径的调节机制的理解，这种复杂的生物途径可以预防癌症和衰老，称为双链断裂修复。这项工作的结果可能有助于开发癌症预防策略和方法，通过预防一些衰老症状来减少老年人的痛苦。

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.