The Role of Small RNAs in Homologous Recombination

小 RNA 在同源重组中的作用

• 批准号: 9167249
• 负责人: JAMES MATTHEW DALEY
• 金额: $ 20.94万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9167249
• 关键词: Address; BRCA1 gene; BRCA2 gene; Base Pairing; Binding; Biochemical; Biological Assay; Bloom Syndrome; Breast; Cancer Etiology; Cell Cycle; Cell Cycle Checkpoint; Cells; Chromosomes; Complementary RNA; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Interstrand Crosslinking; DNA Sequence Alteration; DNA Structure; DNA biosynthesis; DNA damage checkpoint; DNA lesion; Development; Double Strand Break Repair; Enzymes; Event; Excision; Failure; Fanconi' s Anemia; Filament; G2 Phase; Genes; Genetic Materials; Genetic Recombination; Genetic Transcription; Genome; Human; Hybrids; In Vitro; Insecta; Invaded; Lead; Lesion; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Mutation; N-terminal; Nucleoproteins; Oncogenes; Organ; Outcome; Ovary; Pathway interactions; Positioning Attribute; Predisposition; Procedures; Process; Protein Family; Proteins; RNA; RNA Binding; RNA Interference; RNA Processing; Reaction; Research; Research Project Grants; Resistance; Resolution; Role; S Phase; SS DNA BP; Sequence Homologs; Sister; Site; Small RNA; Syndrome; System; Tail; Testing; Tumor Suppressor Genes; Tumor Suppressor Proteins; Work; Yeasts; base; cancer genetics; cancer risk; cancer therapy; ds-DNA; genome-wide; helicase; homologous recombination; insight; malignant breast neoplasm; mutant; novel; novel strategies; presynaptic; recombinase; reconstitution; repaired; research study; tumorigenesis

PROJECT SUMMARY/ABSTRACT DNA double-strand breaks (DSBs) are among the most toxic DNA lesions because failure to repair them accurately can lead to large-scale genome rearrangements. Homologous recombination (HR) is a major, conserved pathway by which cells repair DSBs. It functions by locating a homologous sequence and using it as a template for DNA synthesis, yielding a highly accurate repair outcome. The importance of HR is underscored by the fact that mutations in a number of HR genes, including the tumor suppressors BRCA1 and BRCA2, are associated with increased cancer risk. Rare genetic cancer predisposition syndromes are also associated with aberrant or defective HR. Mutations in the BLM helicase, which participates in the initiation of HR as well as resolution of DNA structures formed late in the process, cause Bloom Syndrome. Fanconi Anemia is caused by mutations in a large complex of “FANC” proteins that detect DNA interstrand crosslinks, lesions that are first excised before being converted into DSBs and routed into the HR pathway. Recent research has uncovered an intriguing link between DSB repair and the RNAi pathway. Specifically, small RNAs have been identified at DSB sites, where they are important for cell cycle checkpoint activation (1) and also HR (2, 3). These RNAs are produced by Dicer and processed by the Argonaute family protein Ago2. Cell-based studies have shown that they are required for the optimal recruitment of RAD51 to DSB sites, but not for upstream steps in HR (2). RAD51 is the recombinase responsible for catalyzing the HR reaction. BRCA2 loads RAD51 onto ssDNA tails formed at the break site, where RAD51 forms a filament that then searches for a homologous DNA template to initiate repair. The mechanism by which small RNAs promote RAD51 loading is unknown. We have developed unique reconstituted systems to examine the steps in HR, including RAD51-ssDNA nucleoprotein filament assembly. In this project, we will apply our expertise to characterize the mechanism by which small RNAs function in HR. In Aim 1, we will purify Ago2, investigate its interaction with RAD51, and generate mutants defective for the interaction. In Aim 2, our unique biochemical systems will be used to pinpoint the role of Ago2 and its associated small RNAs in loading RAD51. The importance of base pairing between the small RNAs and the ssDNA tails onto which RAD51 loads will be assessed, and mutants generated in Aim 1 will be tested. Interplay with DSS1, a BRCA2-interacting protein that assists in removing RPA from the ssDNA to make way for RAD51, will also be investigated. Besides characterizing the novel role of small RNAs in HR, this project will contribute to our mechanistic understanding of a BRCA2-dependent step in HR, and will therefore provide insights into the initial events that lead to tumorigenesis in the breast, ovaries and other organs.

项目摘要/摘要 DNA双链断裂（DSB）是最有毒的DNA病变之一，因为无法修复 它们准确地导致大规模的基因组重排。同类重组（HR）是 细胞修复DSB的主要，保守的途径。它通过定位同源序列来发挥作用 并将其用作DNA合成的模板，得出高度准确的修复结果。这 人力资源的重要性强调了以下事实：许多人力资源基因的突变，包括 肿瘤补充剂BRCA1和BRCA2与癌症风险增加有关。罕见的遗传癌 倾向综合征也与异常或缺陷的HR有关。 BLM中的突变 聚合酶参与人力资源的主动性以及在后期形成的DNA结构的分辨率 该过程，导致Bloom综合征。 Fanconi贫血是由大型复合物中的突变引起的 检测DNA Interstrand交叉链接的“狂法”蛋白质，首先在成为先进的病变 转换为DSB并将其路由到HR途径。 最近的研究发现了DSB修复与RNAi途径之间的有趣联系。 具体而言，在DSB站点已经鉴定出小的RNA，对于细胞周期很重要 检查点激活（1）以及HR（2，3）。这些RNA由DICER生产，并由 Argonaute家庭蛋白AGO2。基于细胞的研究表明，最佳需要它们 将RAD51募集到DSB站点，但没有用于HR（2）中的上游步骤。 RAD51是重组酶 负责催化人力资源反应。 BRCA2将Rad51加载到休息时形成的ssDNA尾巴上 站点，Rad51形成细丝，然后搜索同源DNA模板以启动 维修。小RNA促进RAD51载荷的机制尚不清楚。 我们已经开发了独特的重构系统来检查HR的步骤，包括RAD51-SSDNA 核蛋白细丝组件。在这个项目中，我们将应用我们的专业知识来表征 小RNA在人力资源中功能的机制。在AIM 1中，我们将净化AGO2，调查其 与rad51的相互作用，并产生突变体有缺陷的相互作用。在AIM 2中，我们的独特 生化系统将用于查明AGO2及其相关的小RNA在加载中的作用 RAD51。小rNA和ssDNA尾巴之间的基础配对的重要性 将评估RAD51载荷，并将测试AIM 1中产生的突变体。与DSS1的相互作用 BRCA2相互作用蛋白有助于从ssDNA中取出RPA以使RAD51的空间 也可以调查。除了表征小型RNA在人力资源中的新作用外，该项目还将 有助于我们对HR中BRCA2依赖步骤的机械理解，因此将 提供有关导致乳房，卵巢和其他器官肿瘤发生的最初事件的见解。