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].

资助父母奖的摘要 同源重组（HR）对于维持基因组稳定性至关重要，并且功能 消除DNA双链断裂和染色体病变。人力资源介质中的突变决定了 许多癌症和遗传疾病的病理发展。双链DNA时，启动HR 断裂被切除以产生单链DNA（ssDNA）悬垂，很容易涂覆 并受复制蛋白A（RPA）保护。乳腺癌2型易感性（BRCA2）蛋白 删除和重塑RPA并促进Rad51重组酶的结合的功能，然后催化 链交换并驱动重组。我们的长期目标是获得对 这些人力资源介体之间的ssDNA交接的时间顺序以及这些介体如何竞争 获得ssDNA及其对癌症等疾病的总体贡献。 RPA由多个不同的 DNA结合结构域（DBD），与ssDNA具有高亲和力结合。固有的DNA结合动力学（结合， 假设单个DBD的解离和重塑）决定了何时，何处和如何RPA 功能。数十年来，RPA的四个DBD被归类为高亲和力（DBDS-A＆B），并且 基于ssDNA结合亲和力的实验测量，低亲和力（DBDS-C＆D） 具有DNA复制，修复和重组的RPA机制的形状模型。使用非典型 氨基酸，我们开发了一种基于荧光的方法来捕获单个DBD的动力学 全长蛋白质的上下文。与古典模型相反，我们发现高亲和力DBD是 动态性，并被RPA的修剪核心所吸引。代替这些令人兴奋的RPA发现 机制，我们在这里建议揭示BRCA2介质如何影响RPA-DBD的动力学 促进HR期间Rad51细丝的形成。我们将确定DNA的上下文和类型 HR中遇到的结构会影响RPA-DBD动力学[AIM 1]。我们将研究的重要性 RPA中的调节热点将调节RPA与BRCA2-DSS1复合物之间的相互作用 [AIM 2]。最后，使用BRCA2，RAD51和RAD52的荧光版本，我们将建立 HR期间从RPA到RAD51的ssDNA交接[AIM 3]。