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.

项目总结/摘要 DNA双链断裂（DSB）是最具毒性的DNA损伤之一， 它们可以导致大规模的基因组重排。同源重组（HR）是一种 细胞修复DSB的主要保守途径。它的功能是定位一个同源序列 并将其作为DNA合成的模板，产生高度准确的修复结果。的 HR的重要性是由以下事实强调的：许多HR基因的突变，包括 肿瘤抑制因子BRCA 1和BRCA 2与癌症风险增加有关。罕见遗传性癌症 易感综合征也与异常或有缺陷的HR有关。 解旋酶，参与HR的起始以及晚期形成的DNA结构的分解， 这个过程导致了布鲁姆综合症范可尼贫血是由一个大的复杂的突变引起的， “FANC”蛋白检测DNA链间交联，在被切除前首先切除病变。 转化为DSB并路由到HR途径。 最近的研究发现了DSB修复和RNAi途径之间有趣的联系。 具体地说，已经在DSB位点鉴定了小RNA，在那里它们对细胞周期很重要 检查点激活（1）和HR（2，3）。这些RNA由切丁酶产生，并由 Argonaute家族蛋白Ago 2。基于细胞的研究表明，它们是最佳的 RAD 51被招募到DSB位点，但不用于HR的上游步骤（2）。RAD 51是重组酶 负责催化HR反应。BRCA 2将RAD 51加载到断裂处形成的ssDNA尾部上 位点，其中RAD 51形成细丝，然后搜索同源DNA模板以启动 修复.小RNA促进RAD 51负载的机制尚不清楚。 我们开发了独特的重组系统来检查HR中的步骤，包括RAD 51-ssDNA 核蛋白丝装配在这个项目中，我们将运用我们的专业知识来描述 小RNA在HR中发挥作用的机制。在目的1中，我们将纯化Ago 2，研究其在HR中的作用。 与RAD 51相互作用，并产生相互作用缺陷的突变体。在目标2中，我们独特的 生物化学系统将用于确定Ago 2及其相关的小RNA在负载中的作用。 RAD51。小RNA和ssDNA尾部之间碱基配对的重要性， 将评估RAD 51负载，并将测试在目标1中产生的突变体。与DSS 1的相互作用，a BRCA 2相互作用蛋白，有助于从ssDNA中去除RPA，为RAD 51让路， 也要加以研究。除了表征小RNA在HR中的新作用外，该项目还将 有助于我们对HR中BRCA 2依赖性步骤的机械理解，因此将 提供对导致乳腺、卵巢和其他器官肿瘤发生的初始事件的深入了解。