Mechanisms of RPA, Recombinases, and Mediators in Homologous Recombination

同源重组中 RPA、重组酶和介体的机制

• 批准号: 10589636
• 负责人: Edwin Antony
• 金额: $ 0.98万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-11 至 2022-07-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10589636
• 关键词: A-Form DNA; Affect; Affinity; Amino Acids; Award; BRCA2 Protein; Binding; Cancer Etiology; Cell physiology; Complex; DNA; DNA Binding; DNA Binding Domain; DNA Double Strand Break; DNA Sequence Rearrangement; DNA Structure; DNA biosynthesis; Defect; Disease; Dissociation; Enzymes; Filament; Fluorescence; Funding; Genetic Recombination; Genome Stability; Genomic Instability; Goals; Hereditary Disease; Individual; Length; Lesion; Maintenance; Malignant Neoplasms; Measurement; Mediator of activation protein; Methods; Modeling; Mutation; Parents; Pathologic; Predisposition; Proteins; RAD52 gene; Rad51 recombinase; Research; Resected; Single-Stranded DNA; Therapeutic Intervention; base; cancer type; homologous recombination; malignant breast neoplasm; protein function; recombinase; repaired; replication factor A; trimer core

Abstract of the funded parent award Homologous recombination (HR) is critical for the maintenance of genomic stability, and functions to eliminate DNA double-strand breaks and chromosomal lesions. Mutations in HR mediators dictate the pathological progression of many cancers and hereditary disorders. HR is initiated when a double-stranded DNA break is nucleolytically resected to generate single-stranded DNA (ssDNA) overhangs, which are readily coated and protected by Replication Protein A (RPA). The Breast Cancer Type 2 Susceptibility (BRCA2) protein functions to remove and remodel RPA and promote binding of the RAD51 recombinase, which then catalyzes strand exchange and drives recombination. Our long-term goals are to gain a mechanistic understanding of the temporal sequence of ssDNA handoff between these HR mediators and how these mediators compete for access to ssDNA and its overall contribution to diseases such as cancer. RPA is composed of multiple distinct DNA binding domains (DBDs) and binds with high affinity to ssDNA. The intrinsic DNA binding dynamics (binding, dissociation and remodeling) of individual DBDs are hypothesized to dictate when, where, and how RPA functions. For several decades the four DBDs of RPA have been classified as high affinity (DBDs-A & B) and low affinity (DBDs-C & D) based on experimental measurements of ssDNA binding affinity of isolated DBDs and have shaped models for RPA mechanism in DNA replication, repair and recombination. Using non-canonical amino acids, we developed a fluorescence-based method to capture the dynamics of individual DBDs in the context of the full-length protein. In contrary to classical models, we uncovered that the high-affinity DBDs are dynamic and are outcompeted by the trimerization core of RPA. In lieu of these exciting discoveries of RPA mechanism, we here propose to uncover how the BRCA2 mediator influences the dynamics of RPA-DBDs to promote the formation of the RAD51 filament during HR. We will establish how the context and type of DNA structures that are encountered in HR affect RPA-DBD dynamics [Aim 1]. We will investigate the importance of a regulatory hotspot in RPA that would modulate the interaction between RPA and the BRCA2-DSS1 complex [Aim 2]. Finally, using fluorescent versions of BRCA2, RAD51 and RAD52, we will establish the mechanism of ssDNA handoff from RPA to RAD51 during HR [Aim 3].

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