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.

父母赠款的摘要 单链DNA结合蛋白（SSB）是十九个伴侣SSB Interactome的创始成员。有 理解互动组的作用机理的根本差距，这是第一个原核生物的家族 确定的寡糖/寡核苷酸结合折叠（OB折）基因组监护人。这个差距的持续存在 代表一个重要的问题，因为在填补之前，对基因组稳定性的完整明确理解将是 缺乏。这种理解至关重要，因为在观察基因组监护人家庭成员中存在灾难性后果 用于基因组稳定性。在较高的生物体中，BRCA2的三个OB折叠中的突变最终导致癌症，并且 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。该建议的主要目的是了解SSB如何与 并调节几种伴侣蛋白和本身，以维持基因组完整性。中心假设是两个区域 SSB被称为链接器和ob-fold（SSB和Interactome Partners中）是所有人的关键要素 蛋白质功能的各个方面。拟议研究的理由是，一旦知道SSB的身体和 在功能上与自身及其伴侣互动，对维持基因组所需的事件的更清晰了解 将获得由这个基因组监护人家族介导的诚信。中心假设将通过 追求两个具体的目标：1）确定链接器/OB折叠网络在SSB函数中的作用； 2）确定 SSB相互作用组的作用机理。在第一个目标下，体内和体外结合测定的结合， HDX-MS，蛋白质晶体学，冷冻EM和单分子生物化学将用于确定是否是否是 接头/OB折叠界面是SSB-Partner结合的主要手段。在第二个目标下，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用于了解接头/ob-如何 折叠网络介导了SSB相互作用的功能在DNA复制，重组和修复的动态反应中。 拟议的研究具有创新性，因为采取了组合策略，新颖的单分子方法 使用的以及我们将使用全长蛋白阐明SSB Intervoreome功能的护理。提议 研究很重要，因为它将首次允许开发SSB Interactome功能的清晰图片 在DNA代谢和基因组完整性的维持中。