Recruitment of End-Processing Factors in DSB 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(DSB)修复，这是一个复杂的生物途径家族，用于修复DNA链断裂。DSB修复的一些模式可以完全恢复DNA序列，并且不会导致遗传信息的丢失，但另一些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是同样有效的。在这些更广泛的问题中，有一些更具体的问题，涉及参与某些DSB修复途径而不是其他途径的蛋白质的招募机制。在面包酵母(S.cerevisiae)中，SAW1蛋白可能通过与Rad52蛋白结合，将Rad1-Rad10蛋白复合体招募到DSB位点，但关于这一过程如何发生的生化细节尚不清楚。本项目将详细介绍 SAW1、RAD10和RAD52蛋白在酿酒酵母DSB修复位点上募集的分子基础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酵母，酿酒酵母：1)Saw1和Rad10向DSB募集的模式是否根据DSB位点两侧的非同源DNA序列的长度而改变，2)形成Rad52-SAW1复合体所需的Rad52和SAW1的最小结合结构域，3)在没有酵母Rad52的情况下，人的Rad52是否能被招募到酵母DSB位点，以及4)人Rad52是否能将Saw1招募到酵母DSB位点，以及导致Rad52“链和退火”功能丧失的突变是否会影响Saw1的招募。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，DSB将被特异性地诱导在活酵母细胞中不同染色体上的荧光标记基因座上，并通过聚合荧光信号的荧光成像来监测它们的修复。荧光标记的RAD52、SAW1和RAD10在DSB上的定位将被用来研究DSB两侧不同长度的非同源DNA序列以及RAD52和SAW1基因突变对DSB招募的影响。体外技术将被用来进一步研究Rad52和SAW1蛋白之间的重要相互作用。这些实验将解决有关向DSB站点招募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将人类痛苦降至最低的临床策略非常重要。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，可能会将癌症风险和与衰老相关的疾病降至最低。

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.