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 代谢错误会产生 DNA 损坏。了解纠正此类 DNA 损伤的细胞 DNA 修复机制对于 对抗基因组不稳定性——这是癌症和相关疾病的主要原因。为了纠正此类错误， 双链 DNA 解开，瞬时打开的单链 DNA (ssDNA) 受到保护， 由复制蛋白 A (RPA)（一种高亲和力多结构域酶）包裹。 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。这是如何发生的精确机制 功能特异性的实现却很难解决。为了解决这一知识差距，我们的长期目标 目标是回答以下问题：a) RPA 与超过两打 DNA 处理进行物理交互 酶；这些交互是如何确定并确定优先级的？ b) RPA 以高亲和力 (KD >10-10M）；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？ d) 情况如何 通过翻译后修饰调节 RPA 的 DNA 和蛋白质相互作用活性？为了解决这些 问题，并研究 RPA 在多种其他 DNA 结合酶存在下的动态，我们 已成功开发出一种实验策略，其中单个 DNA 结合域 (DBD) RPA 用荧光团标记。与 ssDNA 结合后，观察到荧光的强烈变化 因此，它充当 DNA 动态的实时报告者。我们通过结合非规范的内容实现了这一目标 氨基酸和使用菌株促进点击化学的荧光团附着。使用这个 通过方法论，我们揭示了 RPA 中的每个域如何在 ssDNA 上结合/解离，并提出了一种新的 RPA 功能范例。 RPA 中有四种 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 研究结果为 DNA 复制、修复和重组中所有 RPA 模型奠定了基础。我们的 在完整的上下文中捕获 RPA 动态的工作揭示了相反的情况，其中 DBD A 和 B 高度 DBD C 和 D 是动态的，而 DBD 是稳定的。这些惊人的发现完全改变了现有的范式 RPA 发挥作用，并构成研究特定 RPA 相互作用蛋白 (RIP) 如何工作的基础 获得 DNA。具体来说，NEIL1 和 UNG2 在碱基切除修复期间进行 RPA 建模（目标 1）和 将研究核苷酸切除修复（目标 2）期间 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（目标 3）。拟议工作的结果将描述 RIP 如何与 RPA 交互、重塑其 DBD 并访问埋藏的 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.