The Role of Small RNAs in Homologous Recombination

小 RNA 在同源重组中的作用

• 批准号: 9329413
• 负责人: JAMES MATTHEW DALEY
• 金额: $ 25.13万
• 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 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9329413
• 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; 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; RAD52 gene; RNA; RNA Binding; RNA Interference; RNA Processing; Rad51 recombinase; Reaction; Recruitment Activity; Research; Research Project Grants; Resistance; Resolution; Role; Route; 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; experimental study; genome-wide; helicase; homologous recombination; insight; malignant breast neoplasm; mutant; novel; novel strategies; presynaptic; recombinase; reconstitution; repaired; 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)是毒性最大的DNA损伤之一，因为无法修复 它们可以准确地导致大规模的基因组重排。同源重组(HR)是一种 主要的，保守的细胞修复DSB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，产生高度准确的修复结果。这个 人力资源的重要性得到了强调，因为许多人力资源基因的突变，包括 肿瘤抑制基因BRCA1和BRCA2与癌症风险增加有关。罕见的遗传癌 易感综合征也与心率异常或缺陷有关。BLM中的突变 解旋酶，它参与HR的启动以及后期形成的DNA结构的解析 这一过程，会导致布鲁姆综合症。范可尼贫血是由一种大型复合体的突变引起的 “FANC”检测DNA链间交联的蛋白质，这种损伤先被切除，然后被切除 转化成DSB并被路由到HR通路。 最近的研究发现了DSB修复和RNAi途径之间的一个有趣的联系。 具体地说，已经在DSB位点发现了小RNA，在那里它们对细胞周期很重要 检查点激活(1)以及HR(2，3)。这些RNA由迪格尔生产，并由 ArgAerte家族蛋白Ago2。基于细胞的研究表明，它们是最佳的 在DSB地点招聘RAD51，但不是在HR(2)的上游步骤。RAD51是重组酶 负责催化HR反应。BRCA2将RAD51加载到断裂时形成的单链DNA尾巴上 RAD51形成丝状结构，然后搜索同源DNA模板以启动 修理。小RNA促进RAD51负载的机制尚不清楚。 我们已经开发了独特的重组系统来检查HR中的步骤，包括RAD51-ssDNA 核蛋白细丝组装。在这个项目中，我们将应用我们的专业知识来描述 小RNA在HR中发挥作用的机制。在目标1中，我们将纯化Ago2，研究其 与RAD51相互作用，并产生对相互作用有缺陷的突变体。在《目标2》中，我们独特的 生化系统将被用来确定Ago2及其相关的小RNA在负荷中的作用 RAD51。小RNA和单链DNA尾部之间碱基配对的重要性 将评估RAD51负载，并测试在AIM 1中产生的突变。与DSS1、 BRCA2-相互作用的蛋白质，帮助从单链DNA中移除RPA，为RAD51让路，Will 也被调查。除了描述小RNA在人力资源中的新作用外，这个项目还将 有助于我们机械地理解人力资源中依赖BRCA2的步骤，并因此 提供对导致乳房、卵巢和其他器官肿瘤发生的初始事件的见解。