Insight into the mechanism of action of the SSB interactome

深入了解 SSB 相互作用组的作用机制

• 批准号: 10610679
• 负责人: Piero R Bianco
• 金额: $ 22.87万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-16 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10610679
• 关键词: BRCA2 gene; Binding; Biochemistry; Biological Assay; Caring; Cryoelectron Microscopy; Crystallography; DNA biosynthesis; DNA metabolism; Defect; Development; Elements; Enzyme Kinetics; Event; Family; Family member; Fostering; Genetic; Genome; Genome Stability; Goals; Health; In Vitro; Lead; Length; Linker-Oligonucleotide; Maintenance; Malignant Neoplasms; Mediating; Microscopy; Mission; Molecular; Mutation; Oligonucleotides; Oligosaccharides; Organism; Proteins; Public Health; Reaction; Research; Resolution; Role; SS DNA BP; Testing; Time; United States National Institutes of Health; combinatorial; genome integrity; human disease; improved; in vivo; innovation; insight; member; novel; parent grant; protein function; public health relevance; recombinational repair; single molecule

ABSTRACT OF THE PARENT GRANT The single-stranded DNA binding protein (SSB) is the founding member of the nineteen-partner SSB interactome. There is a fundamental gap in understanding the mechanism of action of the interactome, the first prokaryotic family of oligosaccharide/oligonucleotide binding fold (OB-fold) genome guardians identified. The continued existence of this gap represents an important problem because, until it is filled, a complete and clear understanding of genome stability will be lacking. This understanding is crucial as defects in OB-fold genome guardian family members have disastrous consequences for genome stability. In higher organisms, mutations in the three OB-folds of BRCA2 ultimately result in cancer, and the proposed studies are therefore directly relevant to human disease. Consequently, the long-term goal is to understand the molecular mechanism of the SSB interactome. The main objective of this proposal is to understand how SSB interacts with, and regulates, several partner proteins and itself, to maintain genome integrity. The central hypothesis is that two regions of SSB known as the linker and the OB-fold, (present in both SSB and interactome partners), are the key elements to all aspects of protein function. The rationale for the proposed research is that once it is known how SSB physically and functionally interacts with both itself and its partners, a clearer understanding of the events required to maintain genome integrity, mediated by this OB-fold family of genome guardians, will be obtained. The central hypothesis will be tested by pursuing two specific aims: 1) determine the role of the linker/OB-fold network in SSB function; and 2) determine the mechanism of action of the SSB interactome. Under the first aim, a combination of in vivo and in vitro binding assays, HDX-MS, protein crystallography, cryo-EM, and single-molecule biochemistry will be used to determine whether the linker/OB-fold interface is the primary means of SSB-partner binding. Under the second aim, enzyme kinetics, genetics, real-time super-resolution microscopy, and single-molecule biochemistry will be used to understand how the linker/OB- fold network mediates SSB interactome function in the dynamic reactions of DNA replication, recombination, and repair. The proposed research is innovative because of the combinatorial strategy taken, the novel single-molecule approaches used, and the care that we will take in elucidating SSB interactome function using full-length proteins. The proposed research is significant because it will allow, for the first time, the development of a clear picture of SSB interactome function in DNA metabolism and the maintenance of genome integrity.

专利授权摘要 单链DNA结合蛋白（SSB）是19对SSB相互作用组的创始成员。有 相互作用组是第一个原核家族，在理解相互作用组的作用机制方面存在根本性差距 寡糖/寡核苷酸结合折叠（OB-折叠）基因组监护人鉴定。这种差距的持续存在 代表了一个重要的问题，因为在它被填满之前，对基因组稳定性的完整和清晰的理解将是 缺乏这种理解是至关重要的，因为OB-折叠基因组监护家庭成员的缺陷会造成灾难性的后果 基因组的稳定性在高等生物体中，BRCA 2的三个OB折叠中的突变最终导致癌症， 因此，拟议的研究与人类疾病直接相关。因此，长期目标是了解 SSB相互作用组的分子机制。本提案的主要目标是了解SSB如何与 并调节几种伴侣蛋白和自身，以维持基因组的完整性。核心假设是两个区域 被称为接头和OB-折叠的SSB（存在于SSB和相互作用组伴侣中）是所有相互作用的关键元件。 蛋白质的功能。拟议研究的基本原理是，一旦知道SSB的物理和 在功能上与自身及其伴侣相互作用，更清楚地了解维持基因组所需的事件， 将获得由该OB折叠基因组监护人家族介导的完整性。中心假设将通过以下方式进行检验： 追求两个具体目标：1）确定接头/OB-折叠网络在SSB功能中的作用;以及2）确定 SSB相互作用组的作用机制。在第一个目标下，结合体内和体外结合测定， HDX-MS、蛋白质晶体学、cryo-EM和单分子生物化学将用于确定 接头/OB-折叠界面是SSB-配偶体结合的主要手段。第二个目标是酶动力学、遗传学， 实时超分辨率显微镜和单分子生物化学将被用来了解如何连接器/OB- 折叠网络在DNA复制、重组和修复的动态反应中介导SSB相互作用体功能。 该研究具有创新性，因为采用了组合策略，新颖的单分子方法 使用，和护理，我们将在阐明SSB的相互作用功能，使用全长蛋白质。拟议 这项研究意义重大，因为它将首次对SSB相互作用组功能有一个清晰的认识 DNA代谢和基因组完整性的维持。