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代谢错误会导致DNA 损坏。对纠正这种DNA损伤的细胞DNA修复机制的了解对于 与基因组不稳定作斗争--这是癌症和相关疾病的主要原因。为了纠正这样的错误， 双链DNA解开，瞬时开放的单链DNA(SsDNA)受到保护， 包被复制蛋白A(RPA)，这是一种高亲和力的多结构域酶。RPA-单链DNA的形成 复合体触发DNA修复检查点反应，是激活大多数DNA修复途径的关键步骤。 与RPA结合的单链DNA被移交给病变特异的DNA修复蛋白。这一过程的确切机制 功能特异性的实现是解决不了的。为了解决这一知识差距，我们的长期计划 目标是回答以下问题：a)RPA与二十多个DNA处理过程进行物理交互 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)与单链DNA结合 &gt；10-10M)；与DNA具有微摩尔亲和力的DNA代谢酶如何去除RPA？C)确实如此 RPA在将招募的酶(以适当的极性)定位到DNA上起到作用吗？D)大家好吗？ 翻译后修饰调节RPA的DNA和蛋白质相互作用活性？要解决这些问题 问题，并研究RPA在多种其他DNA结合酶存在时的动力学，我们 已经成功地开发出一种实验策略，其中单个DNA结合结构域(DBD) RPA用荧光团标记。当与单链DNA结合时，观察到荧光的强烈变化，并 因此，它在DNA上的动态是一个实时记者。我们通过将非规范的 利用菌株的氨基酸和荧光团的附着促进了点击化学。使用这个 方法，我们已经揭示了RPA中的每个结构域如何与单链DNA结合/解离，并提出了一个新的 RPA功能的范例。在RPA中有四个DBD(A、B、C和D)，三十多年来，DBD-A和 根据对分离的DBD的生化研究，B被认为具有最高的亲和力。这些 这些发现为RPA在DNA复制、修复和重组方面的所有模型奠定了基础。我们的 在完整环境中捕获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 RPA的功能并构成拟议工作的基础，该工作研究特定的RPA相互作用蛋白(RIP)如何 获取DNA。具体而言，在碱基切除修复期间由NEIL1和UNG2进行的RPA建模(目标1)和 在核苷酸切除修复过程中的XPA(目标2)将被调查。此外，磷酸化在蛋白合成中的作用 将探索在DNA修复中确定RPA的特异性(目标3)。拟议工作的结果将勾勒出 RIPs如何与RPA相互作用，如何重塑其DBD，以及如何获得埋藏的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.