Molecular Mechanisms Underlying DNA Double-Strand Break and Crosslink Repair

 描述（由申请方提供）：导致自发性和双链断裂诱导的DNA损伤的检测、检查点反应和修复的细胞事件和分子途径尚不完全清楚。虽然在过去几年中已经取得了很大的进步，但重要的修复途径和遗传相互作用仍在探索中。全面了解这一领域的所有遗传相互作用是一个重要的目标，因为DNA损伤修复途径通常在癌细胞中发生遗传改变。事实上，参与这些途径的许多基因本身就是化疗药物的靶点。拟议的工作将通过一系列遗传和细胞生物学实验揭示对DNA复制、重组和修复重要的新型遗传关系和途径。一些实验将通过对DNA损伤敏感突变体进行全面的上位性分析来探索参与双链断裂修复的途径。这些基因之间的上位关系将揭示它们影响哪些途径。来自癌症基因组图谱（TCGA）和METABRIC财团分析的9，000多个肿瘤的基因拷贝数和表达数据用于优先考虑参与DNA复制，重组和修复（3R）的93个基因的过表达研究，这些基因通常在癌症中过表达。与功能丧失突变体相比，基因产物的过表达常常导致不同的遗传失衡。拟议的工作将通过构建基于癌症相关3R基因的过表达遗传相互作用组来模拟3R基因过表达的后果，从而揭示涉及复制，重组和修复的新途径。DNA修复的重要过程是同源重组和寻找同源性。最近的研究表明，染色体在DNA损伤后增加了它们的移动性。我们已经发现，重要的基因和蛋白质，如RecA同源物和重组酶，RAD 51，以及MRX复合物有助于这种效果。通过利用经典的遗传方法，我们将进一步表征这些和其他因素对迁移率和同源性搜索的贡献。我们还将研究DNA末端在双链断裂形成过程中的行为，以解决长期存在的关于这些末端在修复过程中如何表现的问题。这项研究的组合实验将大大增加我们对DNA损伤如何修复的理解。

 DESCRIPTION (provided by applicant): The cellular events and molecular pathways leading to the detection, checkpoint response, and repair of spontaneous and double strand break induced DNA damage are not completely known. Although great strides have been made over the last few years, important repair pathways and genetic interactions are still being uncovered. A full understanding of all the genetic interactions in this area is an important goal, since DNA damage repair pathways are often genetically altered in cancer cells. Indeed, many of the genes involved in these pathways are themselves targets of chemotherapeutic drugs. The proposed work will reveal novel genetic relationships and pathways important for DNA replication, recombination, and repair, through a series of genetic and cell biological experiments. Some experiments will explore the pathways involved in double strand break repair by performing a comprehensive epistasis analysis of DNA damage sensitive mutants. The epistasis relationship amongst these genes will reveal which pathways they affect. Gene copy-number and expression data, from over 9,000 tumors profiled by The Cancer Genome Atlas (TCGA), and METABRIC consortiums, were used to prioritize overexpression studies of 93 genes involved in DNA replication, recombination and repair (3R) that are commonly overexpressed in cancer. Overexpression of gene products often result in different genetic imbalances compared to loss of function mutants. The proposed work will model the consequences of overexpressing 3R genes by constructing an overexpression based genetic interactome for cancer-relevant 3R genes, and thereby uncover novel pathways involved in replication, recombination, and repair. Important processes in DNA repair are homologous recombination and the search for homology. Recent studies have indicated that chromosomes increase their mobility after DNA damage. We have found that important genes and proteins like the RecA homolog and recombinase, RAD51, as well as the MRX complex contribute to this effect. By utilizing classic genetic approaches, we will further characterize the contributions to mobility and homology search of these and other factors. We will also examine the behavior of DNA ends during double-strand break formation to resolve long-standing questions about how these ends behave during repair. The combined experiments of this study will add significantly to our understanding of how DNA lesions are repaired.