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.

摘要