Coordination of DNA Metabolism by Replication Protein A

复制蛋白 A 协调 DNA 代谢

• 批准号: 10623523
• 负责人: Edwin Antony
• 金额: $ 50.12万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623523
• 关键词: Address; Adopted; Affinity; Aging; Amino Acids; Architecture; Binding; Biochemical; Biophysics; Cancer Etiology; Cells; Chromosomal Rearrangement; Complex; DNA; DNA Binding; DNA Binding Domain; DNA Maintenance; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA damage checkpoint; DNA metabolism; Defect; Dissociation; Enzymes; Fluorescence; Gatekeeping; Genetic; Genetic Recombination; Genome; Genome Stability; Goals; Hereditary Disease; Individual; Investigation; Kinetics; Knowledge; Label; Lead; Length; Metabolic; Metabolism; Modeling; Molecular; Nucleosomes; Pathway interactions; Phosphorylation; Positioning Attribute; Post-Translational Protein Processing; Process; Property; Proteins; Replication-Associated Process; Role; Single-Stranded DNA; Site; Specificity; Telomere Maintenance; Therapeutic; Thermodynamics; Work; dexterity; ds-DNA; flexibility; genome integrity; novel; protein function; recruit; repaired; replication factor A; telomere; tool

Summary DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. The precise mechanisms of how RPA imparts functional specificity is poorly resolved. Towards addressing this gap in knowledge, our long-term goals are to answer the following questions: a) RPA physically interacts with over three dozen DNA processing enzymes. How are these interactions determined, regulated, and prioritized? b) RPA binds to ssDNA with high affinity (KD <10-10 M). How do DNA metabolic enzymes that bind to ssDNA with hundred-fold lower affinities remove RPA? c) RPA plays a role in positioning the recruited enzymes (with appropriate polarity) onto the DNA. What are the structural, kinetic, and thermodynamic properties that regulate this process? d) How are the DNA and protein interaction activities of RPA tuned by post translational modifications such as phosphorylation? RPA achieves functional dexterity through a multi-domained architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. This dynamic plasticity has hindered structural, biochemical, and biophysical investigations of full-length RPA. Over the past eight years, our group has developed non-canonical amino acid based site-specific fluorescence labeling tools to investigate the dynamics of the individual domains of RPA. While difficult to accomplish, our breakthrough enabled us to reestablish how the individual domains of RPA bound, dissociated, and remodeled during various DNA metabolic processes. The findings were in stark contrast to commonly assumed models for RPA function and has opened numerous avenues to finally investigate and establish how RPA functions in specific DNA metabolic processes. For example, we showed that the commonly assumed high-affinity DNA binding domains of RPA were in fact the most dynamic and not bound to ssDNA in the context of the full-length protein. Utilizing our powerful biochemical, structural, and biophysical toolkit we here seek to resolve how RPA functions in the context of nucleosomes, R-loops, telomere, and in other DNA repair pathways.

概括 DNA代谢过程包括复制，修复，重组和端粒维持 单链DNA（ssDNA）。在每个复杂过程中，数十个蛋白质在 ssDNA模板。但是，当双链DNA解开时，瞬时开放的ssDNA受到保护 并由高亲和力异三个聚合物ssDNA结合蛋白A（RPA）覆盖。几乎全部下游 DNA过程必须首先重塑/删除RPA或函数，以访问RPA下的SSDNA。 RPA-SSDNA复合物的形成触发DNA损伤检查点响应，是关键步骤 激活大多数DNA修复和重组途径。因此，除了保护暴露的ssDNA外， RPA充当守门人，以定义DNA维持和基因组完整性中的功能特异性。这 RPA如何赋予功能特异性的精确机制无法解决。要解决这个差距 在知识中，我们的长期目标是回答以下问题：a）RPA与过度互动 三打DNA加工酶。这些相互作用如何确定，调节和优先级？ b） RPA与具有高亲和力的ssDNA结合（KD <10-10 m）。如何与ssDNA结合的DNA代谢酶与 较低的亲和力消除了RPA？ c）RPA在定位招募酶（带有 适当的极性）到DNA上。调节的结构，动力学和热力学特性是什么 这个过程？ d）如何通过翻译后调谐RPA的DNA和蛋白质相互作用活性 修饰，例如磷酸化？ RPA通过多域架构实现功能灵活性 利用通过柔性接头连接的几个DNA结合和蛋白质相互作用结构域。这个灵活的 模块化体系结构使RPA能够采用针对特定DNA代谢角色量身定制的无数配置。 这种动态可塑性阻碍了全长RPA的结构，生化和生物物理研究。 在过去的八年中，我们的小组开发了基于非经典氨基酸的位点特异性荧光 标记工具来研究RPA各个域的动力学。虽然很难完成，但我们 突破使我们能够重新建立RPA的各个域如何绑定，解离和重塑 在各种DNA代谢过程中。这些发现与通常假定的模型形成鲜明对比 RPA功能，并开放了许多途径，以最终调查并确定RPA在 特定的DNA代谢过程。例如，我们表明通常假定的高亲和力DNA RPA的结合域实际上是最动态的，在全长的背景下与ssDNA没有结合 蛋白质。利用我们强大的生化，结构和生物物理工具包，我们在这里寻求解决RPA的方式 在核小体，R环，端粒和其他DNA修复途径的背景下起作用。