Mechanisms of DNA hand-off during lesion repair in BER and NER

暴露于环境毒素、辐射和内源性DNA代谢的错误引起DNA 损害细胞DNA修复机制的知识，纠正这种DNA损伤是至关重要的， 与基因组不稳定性作斗争-这是癌症和相关疾病的主要原因。为了纠正这些错误， 双链DNA被解绕，瞬时打开的单链DNA（ssDNA）被保护， 由复制蛋白A（RPA）包被，RPA是一种高亲和力的多结构域酶。RPA-ssDNA的形成 复合物触发DNA修复检查点反应，是激活大多数DNA修复途径的关键步骤。 RPA结合的ssDNA被传递给损伤特异性DNA修复蛋白。这一过程的精确机制 实现的功能特异性很难分辨。为了解决这一知识差距，我们的长期 目标是回答以下问题：a）RPA与二十多个DNA加工过程发生物理相互作用 酶;这些相互作用是如何确定和优先考虑的？B）RPA以高亲和力（KD）结合ssDNA > 10 - 10 M）;以微摩尔亲和力与DNA结合的DNA代谢酶如何去除RPA？（c）是否 RPA在将招募的酶（具有适当的极性）定位到DNA上方面发挥作用？（二）如何 翻译后修饰调节RPA的DNA和蛋白质相互作用活性？解决这些 问题，并研究RPA在多种其他DNA结合酶存在下的动力学，我们 已经成功地开发了一种实验策略，其中单个DNA结合结构域（DBD） RPA用荧光团标记。在与ssDNA结合后，观察到荧光的强烈变化， 因此可以作为其在DNA上动态的实时报告者。我们通过将非经典的 氨基酸和使用菌株促进的点击化学连接荧光团。使用此 方法，我们已经揭示了RPA内的每个结构域如何在ssDNA上结合/解离，并提出了一个新的 RPA功能的范例。RPA中有四种DBD（A、B、C和D），三十多年来，DBD-A & 基于分离的DBD的生物化学研究，认为B以最高亲和力结合。这些 这些发现为RPA在DNA复制、修复和重组中的所有模型奠定了基础。我们 在全长上下文中捕获RPA动态的工作揭示了相反的情况，其中DBD A和B高度 动态的，而DBD C & D是稳定的。这些惊人的发现完全改变了现有的范式， RPA的功能和形式的基础上提出的工作，调查如何特定的RPA相互作用蛋白质（RIP） 获取DNA具体而言，在碱基切除修复（Aim 1）和缺失修复（Aim 2）期间， 将研究核苷酸切除修复（Aim 2）过程中XPA的作用。此外，磷酸化的作用， 将探索确定RPA在DNA修复中的特异性（目的3）。拟议工作的结果将描述 RIP如何与RPA相互作用，重塑其DBD并获得隐藏的ssDNA。

Exposure to environmental toxins, radiation and errors in endogenous DNA metabolism give rise to DNA damage. Knowledge of the cellular DNA repair mechanisms that correct such DNA lesions are vital towards combating genomic instability – a prevailing cause of cancers and associated disorders. To correct such errors, double stranded DNA is unwound and the transiently opened single-stranded DNA (ssDNA) is protected and coated by Replication Protein A (RPA), a high affinity multi-domain enzyme. Formation of RPA-ssDNA complexes trigger the DNA repair checkpoint response and is a key step in activating most DNA repair pathways. ssDNA-bound by RPA is handed-off to lesion-specific DNA repair proteins. The precise mechanisms of how this functional specificity is achieved 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 two dozen DNA processing enzymes; how are these interactions determined and prioritized? b) RPA binds to ssDNA with high affinity (KD >10-10 M); how do DNA metabolic enzymes that bind to DNA with micromolar affinities remove RPA? c) Does RPA play a role in positioning the recruited enzymes (with appropriate polarity) onto the DNA? d) How are the DNA and protein interaction activities of RPA tuned by post translational modifications? To address these questions, and to investigate the dynamics of RPA in the presence of multiple other DNA binding enzymes, we have successfully developed an experimental strategy where the individual DNA binding domains (DBDs) of RPA are labeled with a fluorophore. Upon binding to ssDNA, a robust change in fluorescence is observed and thus serves as a real-time reporter of its dynamics on DNA. We achieved this through incorporation of noncanonical amino acids and attachment of fluorophores using strain promoted click chemistry. Using this methodology, we have uncovered how each domain within RPA binds/dissociates on ssDNA and present a new paradigm for RPA function. There are four DBDs (A, B, C and D) in RPA and, for over three decades, DBD-A & B have been thought to bind with highest affinity based on biochemical investigation of isolated DBDs. These findings have served as a foundation for all models of RPA in DNA replication, repair and recombination. Our work capturing RPA dynamics in the full-length context reveals the opposite, where DBDs A & B are highly dynamic whereas DBDs C & D are stable. These startling findings completely alter the existing paradigm for RPA function and form the basis of the proposed work investigating how specific RPA interacting proteins (RIPs) gain access to DNA. Specifically, RPA modeling by NEIL1 and UNG2 during base excision repair (Aim 1) and by XPA during nucleotide excision repair (Aim 2) will be investigated. In addition, the role of phosphorylation in determining RPA specificity in DNA repair will be explored (Aim 3). Results from the proposed work will delineate how RIPs interact with RPA, remodel its DBDs and gain access to the buried ssDNA.