The Role of Small RNAs in Homologous Recombination

小 RNA 在同源重组中的作用

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

项目概要/摘要 DNA 双链断裂 (DSB) 是毒性最强的 DNA 损伤之一，因为无法修复它们准确地可以导致大规模的基因组重排（HR）。细胞修复 DSB 的主要保守途径，它通过定位同源序列发挥作用。并将其用作 DNA 合成的模板，产生高度准确的修复结果。许多 HR 基因（包括肿瘤抑制因子 BRCA1 和 BRCA2 与罕见遗传性癌症风险增加相关。易感综合征也与 BLM 异常或缺陷相关。解旋酶，参与 HR 的启动以及晚期形成的 DNA 结构的解析这个过程，导致布卢姆综合症是由一个大复合体的突变引起的。 “FANC”蛋白可检测 DNA 链间交联，即在被切除之前首先切除的病变转化为 DSB 并进入 HR 路径。最近的研究发现 DSB 修复和 RNAi 通路之间存在有趣的联系。具体来说，小 RNA 已在 DSB 位点被鉴定，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 Dicer 产生并由 Dicer 加工。基于细胞的研究表明，Argonaute 家族蛋白 Ago2 是实现最佳效果所必需的。将 RAD51 招募到 DSB 位点，但不用于 HR 中的上游步骤 (2)。负责催化 HR 反应，BRCA2 将 RAD51 加载到断裂处形成的 ssDNA 尾部上。 RAD51 在此形成细丝，然后搜索同源 DNA 模板以启动小RNA促进RAD51加载的机制尚不清楚。我们开发了独特的重组系统来检查 HR 中的步骤，包括 RAD51-ssDNA 在这个项目中，我们将运用我们的专业知识来表征核蛋白丝组装。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，并产生相互作用缺陷的突变体，在目标 2 中，我们独特的。生化系统将用于查明 Ago2 及其相关小 RNA 在加载中的作用 RAD51。小 RNA 和 ssDNA 尾部之间碱基配对的重要性将评估 RAD51 负载，并测试 Aim 1 中生成的突变体与 DSS1 的相互作用。 BRCA2 相互作用蛋白有助于从 ssDNA 中去除 RPA，为 RAD51 让路，除了表征小 RNA 在 HR 中的新作用外，该项目还将进行研究。有助于我们对 HR 中 BRCA2 依赖性步骤的机制理解，因此将提供对导致乳房、卵巢和其他器官肿瘤发生的初始事件的见解。