Coordination of DNA Metabolism by Replication Protein A

摘要 DNA代谢过程包括复制、修复、重组和端粒维持 单链DNA(SsDNA)。在这些复杂的过程中，几十种蛋白质共同作用于 单链DNA模板。然而，当双链dna解开时，暂时开放的ssdna受到保护。 并包被高亲和力的异三聚体单链DNA结合复制蛋白A(RPA)。几乎所有的下游 DNA过程必须首先重塑/移除RPA或同时运行，才能访问RPA下遮挡的单链DNA。 RPA-ssDNA复合体的形成触发了DNA损伤检查点反应，是 激活大多数DNA修复和重组途径。因此，除了保护暴露的单链DNA外， RPA在DNA维护和基因组完整性中起到把关作用，定义功能特异性。这个 RPA如何赋予功能特异性的确切机制尚未得到很好的解决。朝着解决这一差距的方向 在知识方面，我们的长期目标是回答以下问题：a)RPA与Over 三打DNA处理酶。如何确定、管理和确定这些交互作用的优先级？b) RPA以高亲和力与单链DNA结合(Kd&lt；10-10M)。DNA代谢酶是如何与单链DNA结合的 亲和力降低数百倍会移除RPA吗？C)RPA在定位招募的酶方面发挥作用(与 适当的极性)连接到DNA上。什么是结构，动力学和热力学性质，调节 这个过程？D)翻译后如何调节RPA的DNA和蛋白质相互作用活性 修饰，如磷酸化？RPA通过多域体系结构实现功能灵活性 利用由柔性连接物连接的几个DNA结合和蛋白质相互作用结构域。这一灵活且 模块化体系结构使RPA能够采用各种针对特定DNA代谢角色量身定做的配置。 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。 在过去的八年里，我们小组开发了基于非规范氨基酸的位置特异性荧光 用于研究RPA各个域的动态的标记工具。虽然很难实现，但我们的 突破性进展使我们能够重新确定RPA的各个域如何绑定、分离和重构 在各种DNA代谢过程中。这一发现与通常假设的模型形成了鲜明对比 RPA的功能，并开辟了许多途径来最终调查和确定RPA在 特定的DNA代谢过程。例如，我们证明了通常认为的高亲和力DNA RPA的结合域实际上是最动态的，并且在全长的上下文中不与ssDNA结合 蛋白。利用我们强大的生化、结构和生物物理工具包，我们在此寻求解决RPA如何 在核小体、R环、端粒和其他DNA修复途径中发挥作用。

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.