Gene conversion and end processing mechanisms during double-strand break repair

描述（由申请人提供）：我的主要研究目标是研究细胞如何对DNA损伤做出反应。准确和有效的修复是保持基因组完整性的必要条件；这种完整性的丧失可导致突变、人类疾病和肿瘤发生。我特别感兴趣的是研究一种特别有害的损伤——DNA双链断裂（DSB）的同源重组（HR）修复机制。在各种模式生物中进行的大量研究有助于目前对HR修复的理解。然而，许多悬而未决的问题使我们无法完全理解HR的机制以及哺乳动物系统中DNA在DSB位点的末端是如何加工的。将采取双重方法来解决这两个问题。首先，利用遗传学方法，研究与dsb的HR修复相关的基因转换机制。有必要对本实验室和其他实验室先前报道的基因转换研究进行扩展。一种新的修复基质将允许更精细地绘制基因转换轨迹。此外，分析错配修复缺陷突变体中的修复事件将发现本应修复的异双工DNA （hDNA）。这种遗传背景可以用作测试以下关于基因转换机制的持久问题的工具：单端与双端链入侵，供体序列的命运，以及与长基因转换束相关的dna与间隙修复。其次，将完成对DNA末端如何在DSB位点加工以及HR产物如何在哺乳动物细胞中形成的物理分析。该分析将包括确定DSB形成的动力学，测量DSB位点的DNA降解率，分析修复所需的蛋白质在DSB上的定位，以及HR修复产物何时以及如何形成。总之，这些研究将阐明基因转换的机制，规范的DSBR模型和SDSA在有丝分裂基因转换修复产物形成中的贡献，并阐明DSB末端加工的机制。

DESCRIPTION (provided by applicant): My broad research goal is to investigate how cells respond to DNA damage. Accurate and efficient repair is necessary to maintain genomic integrity; loss of this integrity can lead to mutagenesis, human disease, and tumorigenesis. In particular, I am interested in studying the mechanisms of homologous recombination (HR) repair of a particularly deleterious lesion, the DNA double-strand break (DSB). Numerous studies in a variety of model organisms have contributed to the current understanding of HR repair. However, many outstanding questions preclude us from completely understanding the mechanisms of HR and how DNA ends at the site of the DSB are processed in mammalian systems. A two-fold approach will be taken to address these both of these questions. First, using genetic approaches, the mechanisms of gene conversion associated with HR repair of DSBs will be investigated. It is necessary to expand on the gene conversion studies previously reported in this lab and others. A novel repair substrate will allow to more finely map gene conversion tracks. Additionally, analyzing repair events in a mismatch repair defective mutant will uncover heteroduplex DNA (hDNA) that would have otherwise been repaired. This genetic background can be used as a tool to test the following persistent questions regarding mechanisms of gene conversion: one-ended vs. two-ended strand invasion, fate of donor sequence, and hDNA vs. gap repair associated with long gene conversion tracts. Second, a physical analysis of how DNA ends are processed at the site of a DSB and how HR products are formed in mammalian cells will be completed. This analysis will include determining the kinetics of DSB formation, measure rates of DNA degradation at the DSB site, analyze of localization of proteins to the DSB that are required for repair, and lastly, when and how HR repair products are formed. Together, these studies will elucidate the mechanisms of gene conversion, the contribution of the canonical DSBR model and SDSA in the formation of mitotic gene conversion repair products, and clarify the mechanism of end processing of a DSB. The ability for a cell to respond to DNA damage is necessary to maintain genomic integrity. A particularly toxic lesion, DSBs, can arise from environmental factors as well as during normal cellular processes. As evident in the numerous diseases, genome instability, tumorigenesis, and mutagenesis associated with inefficient or inaccurate repair of DSBs, it is integral to understand the mechanisms required in repairing these deleterious lesions.