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].

资助家长奖摘要 同源重组 (HR) 对于维持基因组稳定性至关重要，其功能 消除DNA双链断裂和染色体损伤。人力资源调解者的突变决定了 许多癌症和遗传性疾病的病理进展。当双链 DNA 形成时 HR 启动 断裂被核酸切除以产生单链 DNA (ssDNA) 突出端，这些突出端很容易被包被 并受到复制蛋白 A (RPA) 的保护。 2 型乳腺癌易感性 (BRCA2) 蛋白 功能是去除和重塑 RPA 并促进 RAD51 重组酶的结合，然后催化 链交换并驱动重组。我们的长期目标是获得对 这些 HR 介体之间 ssDNA 交接的时间顺序以及这些介体如何竞争 单链 DNA 的获取及其对癌症等疾病的总体贡献。 RPA 由多个不同的 DNA 结合域 (DBD) 并以高亲和力与 ssDNA 结合。内在的 DNA 结合动力学（结合， 假设单个 DBD 的解离和重塑决定 RPA 的时间、地点和方式 功能。几十年来，RPA 的四种 DBD 已被归类为高亲和力 (DBD-A 和 B) 和 低亲和力 (DBD-C & D) 基于分离 DBD 的 ssDNA 结合亲和力的实验测量 形成了 DNA 复制、修复和重组中 RPA 机制的模型。使用非规范 氨基酸，我们开发了一种基于荧光的方法来捕获单个 DBD 的动态 全长蛋白质的背景。与经典模型相反，我们发现高亲和力 DBD 是 动态且被 RPA 的三聚核心击败。代替 RPA 的这些令人兴奋的发现 机制，我们在这里建议揭示 BRCA2 介质如何影响 RPA-DBD 的动态 促进 HR 期间 RAD51 丝的形成。我们将确定 DNA 的背景和类型 HR 中遇到的结构会影响 RPA-DBD 动态 [目标 1]。我们将调查其重要性 RPA 中的一个调控热点，可调节 RPA 和 BRCA2-DSS1 复合物之间的相互作用 [目标 2]。最后，使用 BRCA2、RAD51 和 RAD52 的荧光版本，我们将建立以下机制： HR 期间 ssDNA 从 RPA 切换到 RAD51 [目标 3]。