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

描述(由申请者提供)：我的主要研究目标是研究细胞对DNA损伤的反应。准确和有效的修复对于保持基因组的完整性是必要的；这种完整性的丧失可能导致突变、人类疾病和肿瘤形成。特别是，我对研究同源重组(HR)修复一种特别有害的损伤-DNA双链断裂(DSB)的机制感兴趣。在各种模式生物中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阻碍了我们完全理解HR的机制，以及DNA在DSB位点的末端是如何在哺乳动物系统中处理的。为了解决这两个问题，我们将采取两种方法。首先，利用遗传学方法，研究与DSB 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以前报道的基因转化研究进行扩展。一种新的修复底物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揭示本来可以修复的异源双链DNA(HDNA)。这一遗传背景可以作为一种工具来测试以下关于基因转换机制的长期存在的问题：单端和双端链入侵，供体序列的命运，以及与长基因转换区相关的hDNA和缺口修复。其次，将完成对DNA末端在DSB位置如何处理以及HR产物如何在哺乳动物细胞中形成的物理分析。这项分析将包括确定DSB形成的动力学，测量DSB部位的DNA降解率，分析修复所需的蛋白质在DSB上的定位，以及HR修复产物的形成时间和方式。综上所述，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的形成中的贡献，并阐明DSB的末端加工机制。 细胞对DNA损伤做出反应的能力是维持基因组完整性所必需的。一种特别有毒的损伤，DSB，可以由环境因素以及在正常的细胞过程中引起。正如与DSB低效或不准确修复相关的许多疾病、基因组不稳定、肿瘤发生和突变所明显的那样，了解修复这些有害损伤所需的机制是不可或缺的。

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.