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: 10015322
• 负责人: Edwin Antony
• 金额: $ 30.3万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-10 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10015322
• 关键词: A-Form DNA; Affect; Affinity; Amino Acids; BRCA2 Protein; Binding; Cancer Etiology; Cell division; Cell physiology; Cells; Complex; DNA; DNA Binding; DNA Binding Domain; DNA Double Strand Break; DNA Sequence Rearrangement; DNA Structure; DNA biosynthesis; DNA replication fork; Defect; Diffusion; Disease; Dissociation; Enzymes; Filament; Fluorescence; Genetic Diseases; Genetic Recombination; Genome Stability; Genomic Instability; Goals; Hereditary Disease; Individual; Ionizing radiation; Kinetics; Label; Lead; Length; Lesion; Maintenance; Malignant Neoplasms; Measurement; Mediator of activation protein; Methodology; Methods; Mitotic; Modeling; Modification; Molecular Conformation; Mutagenesis; Mutation; Nucleic Acid Regulatory Sequences; Nucleoproteins; Pathologic; Pathway interactions; Phosphorylation Site; Phosphoserine; Post-Translational Protein Processing; Predisposition; Property; Proteins; RAD52 gene; Rad51 recombinase; Replication Error; Research; Resected; Resolution; Role; Single-Stranded DNA; Slide; Stretching; Technology; Therapeutic Intervention; base; cancer genetics; cancer type; experimental study; flexibility; homologous recombination; insight; malignant breast neoplasm; novel; protein function; protein protein interaction; recombinase; repaired; replication factor A; single molecule; tool; trimer core

PROJECT SUMMARY 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 be 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].

项目摘要 同源重组（HR）对于维持基因组稳定性至关重要，其功能是 消除DNA双链断裂和染色体损伤。HR介导因子的突变决定了 许多癌症和遗传性疾病的病理进展。当双链DNA 断裂被核裂解切除以产生单链DNA（ssDNA）突出端，其易于被 并受复制蛋白A（RPA）保护。乳腺癌2型易感性（BRCA 2）蛋白 其功能是去除和重塑RPA，并促进RAD 51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一个机械的理解， 这些HR介体之间ssDNA切换的时间序列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），并以高亲和力结合ssDNA。固有的DNA结合动力学（结合， 假设个体DBD的解离和重塑）决定RPA的时间、地点和方式 功能协调发展的几十年来，RPA的四种DBD已被分类为高亲和力（DBDs-A & B）和低亲和力（DBDs-B）。 基于分离的DBD的ssDNA结合亲和力的实验测量， 已经形成了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 动态的，并被RPA的三聚核心所击败。代替RPA的这些令人兴奋的发现 机制，我们在这里提出揭示如何BRCA 2调解人影响的RPA-DBDs的动态， 在HR过程中促进RAD 51细丝的形成。我们将建立DNA的背景和类型如何在HR过程中促进RAD 51细丝的形成。 在HR中遇到的结构影响RPA-DBD动力学[目标1]。我们将研究 RPA中的一个调控热点，可调节RPA与BRCA 2-DSS 1复合物之间的相互作用 [Aim 2]。最后，使用BRCA 2，RAD 51和RAD 52的荧光版本，我们将建立 在HR期间ssDNA从RPA切换到RAD 51 [目的3]。