Fluorescent probes to monitor Rad51 nucleoprotein dynamics

用于监测 Rad51 核蛋白动态的荧光探针

基本信息

批准号：
9108113
负责人：
Edwin Antony
金额：
$ 30.48万
依托单位：
MARQUETTE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9108113
关键词：
Address Affect Amino Acids Architecture Area BRCA2 gene Binding Binding Proteins Biological Assay Bloom Syndrome Cells Chromosome Pairing Chromosomes Complex Complication DNA Binding DNA Double Strand Break Data Defect Development Disease Dissociation Down Syndrome Event Excision Filament Fluorescence Fluorescent Probes Genomic Instability Goals Growth Health Hereditary Disease Investigation Kinetics Lead Malignant Neoplasms Malignant neoplasm of ovary Measures Mediating Mediator of activation protein Modeling Molecular Monitor Mutagenesis Mutation Nucleoproteins Pathway interactions Process Proteins Rad51 recombinase Reaction Regulation Reporter Research Research Project Grants Resected Resolution Role SS DNA BP Signal Transduction Single-Stranded DNA Site Structure System Time Universities Utah Werner Syndrome base biochemical tools cancer genetics ds-DNA fluorophore functional outcomes helicase homologous recombination malignant breast neoplasm mutant protein protein interaction recombinase repaired response spatiotemporal tool undergraduate student

项目摘要

DESCRIPTION (provided by applicant): Double strand DNA breaks in the cell are repaired primarily by the Homologous Recombination (HR) system. Defective or unregulated HR results in chromosome rearrangements leading to genomic instability, which in turn results in a variety of cancers and hereditary disorders. Formation of the Rad51 filament on single- stranded DNA (ssDNA) is one of the key events in the HR pathway and is under tight regulation by both pro- and anti-recombinogenic mediator proteins. Our long-term goal is to precisely determine the dynamics of Rad51 nucleoprotein filament formation and its disassembly on ssDNA and the mechanism by which various pro- and anti-HR mediator proteins alter these dynamics to influence HR. Technical limitations have hindered the study of Rad51 nucleoprotein filament formation on single-stranded DNA (ssDNA). We propose to overcome these obstacles through the development of fluorescent Rad51 and RPA probes which, when bound to ssDNA will undergo a change in fluorescence. We will be utilizing the unnatural amino acid incorporation strategy to develop these probes which will be combined with stopped flow kinetic assays to monitor the dynamics of the nucleoprotein filament in real-time. Since our probes are direct reporters of Rad51/RPA association/dissociation, we will utilize this fluorescent tool-kit to investigate how Rad51 and RPA bind to ssDNA when they are both present in a multi-protein experimental setup. Significance of studying Rad51 filament formation on both ssDNA and dsDNA substrates is highlighted by our recent findings that the bound-DNA context of a Rad51 filament diametrically controls the activity of Srs2. Srs2 is an anti-recombinase that clears Rad51 nucleoprotein filaments from ssDNA and as a helicase is capable of unwinding dsDNA. Physical interaction between Rad51 and Srs2 is required to dismantle the nucleoprotein filament and is observed strictly on ssDNA. When Rad51 is bound on dsDNA, it inhibits the DNA unwinding activity of Srs2 through the very same physical interaction interface. The Specific Aims are to: (1) Develop a real-time fluorescence-based tool kit to measure nucleoprotein filament dynamics on ssDNA. (2) Determine the mechanism behind the DNA-context dependent regulation of Srs2 activity by Rad51. We will obtain kinetic parameters for the various steps in the nucleoprotein assembly and disassembly process and uncover the molecular details of the interaction between Srs2 and Rad51. These studies will enable us to build a precise mechanistic model of how Rad51 nucleoprotein filaments are formed and how mediator proteins alter its dynamics. This information is critical in understanding the role of mediator proteins in HR associated defects, and to uncover why mutations in these proteins lead to genomic instability, cancers and associated genetic disorders. In addition to addressing these research questions, we will develop multiple research projects well suited to engage undergraduate students in bio-medically relevant research at Utah State University.

描述（由申请人提供）：细胞中的双链 DNA 断裂主要由同源重组 (HR) 系统修复。有缺陷或不受调节的 HR 会导致染色体重排，从而导致基因组不稳定，进而导致多种癌症和遗传性疾病。单链 DNA (ssDNA) 上 Rad51 丝的形成是 HR 通路中的关键事件之一，并且受到亲和蛋白的严格调控。我们的长期目标是精确确定 Rad51 核蛋白丝形成及其在 ssDNA 上分解的动力学，以及各种亲和抗 HR 介导蛋白改变这些动力学以影响 HR 的机制。阻碍了 Rad51 核蛋白丝在单链 DNA (ssDNA) 上形成的研究，我们建议通过开发荧光 Rad51 和 RPA 探针来克服这些障碍。当与 ssDNA 结合时，我们将利用非天然氨基酸掺入策略来开发这些探针，并将其与停流动力学分析相结合，以实时监测核蛋白丝的动态。我们的探针是 Rad51/RPA 结合/解离的直接产生者，我们将利用该荧光工具包来研究当 Rad51 和 RPA 都存在于多蛋白实验中时，它们如何与 ssDNA 结合我们最近的发现强调了研究 Rad51 丝在 ssDNA 和 dsDNA 底物上形成的重要性，即 Rad51 丝的结合 DNA 背景直接控制 Srs2 的活性，Srs2 是一种从 ssDNA 中清除 Rad51 核蛋白丝的抗重组酶。并且由于解旋酶能够解开 dsDNA，所以 Rad51 和 Srs2 之间需要物理相互作用。拆除核蛋白丝并在 ssDNA 上严格观察。当 Rad51 与 dsDNA 结合时，它通过完全相同的物理相互作用界面抑制 Srs2 的 DNA 解旋活性。具体目标是： (1) 开发实时荧光。基于工具套件，用于测量 ssDNA 上的核蛋白丝动力学 (2) 通过以下方式确定 Srs2 活性的 DNA 背景依赖性调节背后的机制。我们将获得核蛋白组装和分解过程中各个步骤的动力学参数，并揭示 Srs2 和 Rad51 之间相互作用的分子细节，这些研究将使我们能够建立 Rad51 核蛋白丝如何形成和的精确机制模型。这些信息对于理解介质蛋白在 HR 相关缺陷中的作用以及揭示为什么这些蛋白质的突变导致基因组不稳定、癌症和相关遗传至关重要。除了解决这些研究问题外，我们还将开发多个非常适合犹他州立大学本科生参与生物医学相关研究的研究项目。