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.

总结 DNA代谢过程包括复制、修复、重组和端粒维持， 单链DNA（ssDNA）。在每一个复杂的过程中，几十种蛋白质在细胞膜上共同发挥作用。 ssDNA模板。然而，当双链DNA解绕时，瞬时打开的ssDNA被保护 并被结合复制蛋白A（RPA）的高亲和力异源三聚体ssDNA包被。几乎所有下游 DNA过程必须首先重塑/去除RPA或同时发挥作用以获取RPA下封闭的ssDNA。 RPA-ssDNA复合物的形成触发DNA损伤检查点反应，并且是DNA损伤的关键步骤。 激活大多数DNA修复和重组途径。因此，除了保护暴露的ssDNA之外， RPA作为守门人的功能，以定义DNA维持和基因组完整性的功能特异性。的 RPA如何赋予功能特异性的精确机制尚不清楚。为了弥补这一差距， 在知识方面，我们的长期目标是回答以下问题：a）RPA与超过 三打DNA加工酶这些互动是如何确定、管理和优先考虑的？B） RPA以高亲和力（KD <10-10 M）结合ssDNA。DNA代谢酶如何与ssDNA结合， 亲和力降低100倍就能去除RPA吗c）RPA在定位所募集的酶中起作用（具有 合适的极性）上。什么是结构，动力学和热力学性质， 这个过程？d）RPA的DNA和蛋白质相互作用活性如何通过翻译后调节 如磷酸化修饰？RPA通过多域架构实现功能灵活性 利用由柔性接头连接的几个DNA结合和蛋白质相互作用结构域。这种灵活的， 模块化架构使RPA能够采用为特定DNA代谢作用量身定制的无数配置。 这种动态可塑性阻碍了结构，生化和生物物理研究全长RPA。 在过去的八年里，我们的小组已经开发了基于非典型氨基酸的位点特异性荧光 标签工具来调查RPA的各个域的动态。虽然很难实现，但我们 这一突破使我们能够重新建立RPA的各个结构域如何结合、解离和重塑 在不同的DNA代谢过程中。研究结果与通常假设的模型形成鲜明对比， RPA的功能，并开辟了许多途径，最终调查和建立如何RPA的功能， 特定的DNA代谢过程例如，我们发现，通常假设的高亲和力DNA RPA的结合结构域实际上是最动态的，并且在全长的情况下不与ssDNA结合。 蛋白利用我们强大的生物化学，结构和生物物理工具包，我们在这里寻求解决如何RPA 在核小体、R环、端粒和其他DNA修复途径中发挥作用。