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: 10574583
• 负责人: Ram I. Mahato
• 金额: $ 31.19万
• 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/10574583
• 关键词: Affinity; BRCA2 gene; Binding; Binding Proteins; Biochemistry; Biological Assay; Biological Models; Biology; C-terminal binding protein; Caring; Cell Death; Complex; Creativeness; Cryoelectron Microscopy; Crystallography; DNA; DNA Binding; DNA biosynthesis; DNA metabolism; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Data; Defect; Development; Disparate; Elements; Enzyme Kinetics; Escherichia coli; Eukaryota; Event; Family; Family member; Fostering; Genetic; Genetic Recombination; Genome; Genome Stability; Goals; Health; In Vitro; Kinetics; Lead; Length; Maintenance; Malignant Neoplasms; Mediating; Mission; Molecular; Multiple Partners; Mutation; Oligonucleotides; Oligosaccharides; Organism; Outcome; Play; Prokaryotic Cells; Proteins; Public Health; Publishing; Reaction; Replication-Associated Process; Research; Role; SS DNA BP; Single-Stranded DNA; Structure; System; Tail; Testing; Time; United States National Institutes of Health; Work; combinatorial; genome integrity; helicase; human disease; improved; in vivo; innovation; insight; member; novel; protein B; protein function; public health relevance; repaired; single molecule; superresolution microscopy

ABSTRACT 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.

抽象的 单链 DNA 结合蛋白 (SSB) 是十九个伙伴 SSB 相互作用组的创始成员。有 在理解相互作用组（第一个原核生物家族）的作用机制方面存在根本差距 鉴定出寡糖/寡核苷酸结合折叠（OB折叠）基因组守护者。这种差距的持续存在 代表了一个重要的问题，因为在它被填补之前，对基因组稳定性的完整和清晰的理解将是 缺乏。这种理解至关重要，因为 OB 折叠基因组监护家庭成员的缺陷会带来灾难性的后果 为了基因组的稳定性。在高等生物中，BRCA2 的三个 OB 折叠的突变最终会导致癌症，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。该提案的主要目标是了解 SSB 如何与以下对象交互： 并调节一些伙伴蛋白及其自身，以维持基因组完整性。中心假设是两个区域 称为连接子的 SSB 和 OB 折叠（存在于 SSB 和相互作用组伙伴中）是所有 蛋白质功能的各个方面。拟议研究的基本原理是，一旦了解 SSB 的物理和 与自身及其伙伴进行功能性相互作用，更清楚地了解维持基因组所需的事件 由基因组守护者的 OB 折叠家族介导的完整性将被获得。中心假设将被检验 追求两个具体目标：1）确定连接子/OB折叠网络在SSB功能中的作用； 2) 确定 SSB 相互作用组的作用机制。第一个目标是结合体内和体外结合测定， HDX-MS、蛋白质晶体学、冷冻电镜和单分子生物化学将用于确定是否 连接子/OB折叠界面是SSB-伙伴结合的主要方式。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 相互作用组功能。 由于所采取的组合策略、新颖的单分子方法，所提出的研究具有创新性 以及我们在使用全长蛋白质阐明 SSB 相互作用组功能时所采取的谨慎态度。拟议的 研究意义重大，因为它将首次使我们能够清晰地了解 SSB 相互作用组功能 DNA 代谢和基因组完整性的维护。