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

BER 和 NER 损伤修复过程中 DNA 传递的机制

• 批准号: 10377257
• 负责人: Edwin Antony
• 金额: $ 0.98万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-06 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377257
• 关键词: Address; Affinity; Amino Acids; Base Excision Repairs; Binding; Biochemical; Cancer Etiology; Cells; Chemistry; Complex; DNA; DNA Binding; DNA Binding Domain; DNA Damage; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA biosynthesis; DNA lesion; DNA metabolism; DNA-Protein Interaction; Defect; Disease; Environmental Carcinogens; Enzyme Interaction; Enzymes; Exposure to; Fluorescence; Foundations; Genetic Recombination; Genomic Instability; Goals; Hand; Hereditary Disease; Individual; Investigation; Knowledge; Label; Length; Lesion; Malignant Neoplasms; Metabolic; Methodology; Modeling; Mutation; Nucleotide Excision Repair; Phosphorylation; Play; Positioning Attribute; Post-Translational Protein Processing; Proteins; Radiation; Reporter; Research; Role; Single-Stranded DNA; Specificity; Therapeutic Intervention; Time; Toxic Environmental Substances; Work; XPA gene; base; ds-DNA; fluorophore; recruit; repaired; replication factor A; response

SUMMARY 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 non- canonical 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 six distinct subdomains (A - F) 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 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.

概括 暴露于环境毒素，辐射和内源性DNA代谢中的误差会引起DNA 损害。了解纠正此类DNA病变的细胞DNA修复机制至关重要 打击基因组不稳定性 - 癌症和相关疾病的主要原因。要纠正此类错误， 双链DNA已解开，瞬时打开的单链DNA（ssDNA）受到保护，并受到保护，并且 通过复制蛋白A（RPA）涂层，一种高亲和力多域酶。 RPA-SSDNA的形成 复合物触发DNA修复检查点响应，是激活大多数DNA修复途径的关键步骤。 通过RPA结合ssDNA已交给病变特异性DNA修复蛋白。如何确切机制 实现功能特异性的解决方案很差。为了解决知识中的这一差距，我们的长期 目标是回答以下问题：a）RPA与超过两打DNA处理进行物理互动 酶；这些相互作用如何确定和优先级？ b）RPA与高亲和力（KD）的ssDNA结合 > 10-10 m）;与微摩尔亲和力与DNA结合的DNA代谢酶如何消除RPA？ c）做 RPA在将招募的酶（具有适当的极性）定位到DNA中发挥作用？ d）如何 RPA的DNA和蛋白质相互作用活性是通过翻译后修饰调整的？解决这些 问题，并在存在多种其他DNA结合酶的情况下研究RPA的动力学，我们 已经成功制定了一种实验策略，其中单个DNA结合域（DBD）的 RPA用荧光团标记。与ssDNA结合后，观察到荧光的稳健变化，并 这是其在DNA上动态的实时记者。我们通过感染非 - 典型的氨基酸和荧光团的附着，使用菌株促进了点击化学。使用此 方法论，我们发现了RPA中的每个域如何在ssDNA上结合/解离并呈现一个新的 RPA功能的范例。 RPA中有六个不同的子域（A -f），在三十多年来，DBD- 基于对孤立DBD的生化研究，A＆B被认为具有最高亲和力的结合。这些 调查结果已成为DNA复制，修复和重组中所有RPA模型的基础。我们的 在全长上下文中捕获RPA动力学的工作揭示了相反，其中DBDS A＆B高度 动态，而DBDS C＆D稳定。这些开始发现改变了RPA功能的现有范例 并构成了拟议工作的基础，研究了特定的RPA相互作用蛋白（RIP）如何获得访问 到DNA。具体而言，基本惊喜维修期间由Neil1和UNG2建模（AIM 1）以及XPA在 将研究核苷酸切除修复（AIM 2）。另外，磷酸化在确定RPA中的作用 将探索DNA修复中的特异性（AIM 3）。拟议工作的结果将描述撕裂如何相互作用 使用RPA，重塑其DBD，并可以访问内置的ssDNA。