Recruitment of End-Processing Factors in DSB 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（DSB）修复，这是一个复杂的修复DNA链断裂的生物途径家族。一些DSB修复模式可以进行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，因为它们在不丢失遗传信息的情况下修复染色体并不都是同样有效的。在这些更广泛的问题中，更具体的问题是关于参与某些DSB修复途径而不参与其他途径的蛋白质募集机制。在面包酵母（酿酒酵母）中，Saw1蛋白将Rad1-Rad10蛋白复合物招募到DSB位点，可能是通过与Rad52蛋白结合，但关于这一过程的生化细节尚不清楚。本项目将详细介绍

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.