Repair of Oxidative Damage in G-Quadruplex Promoter DNA

项目摘要/摘要 环境暴露和生活方式的选择会导致细胞氧化应激， 以产生大量的活性氧(ROS)为特征的。ROS Wreak 对DNA碱基结构的破坏，鸟嘌呤修饰产生损伤8-氧-7，8- 二氢鸟嘌呤(8oxoG)特别流行。如果不修复，8oxoG就会发生突变，导致 可以引发和促进人类疾病的G到T颠倒突变。富含鸟嘌呤的G- 四链(G4)形成序列在基因组的启动子近端富含， 使这些区域成为8oxoG病变的热点。8oxoG根部切除修补术 G4启动子序列上的(BER)途径(即，血管内皮生长因子)可以调节转录，但 G4启动子内修复活性的分子水平相互作用和机制细节 上下文没有被很好地理解。拟议研究的总体目标是描述 8oxoG与基因修复的分子水平相互作用及配位事件 氧化应激反应中血管内皮生长因子启动子的增强。建议进行的实验 为解决这一问题，将分两个阶段进行。在最初指导的K99阶段，X射线 结晶学，先进的核酸动力学，单分子荧光，以及人类细胞- 基于转录的测试将被用来表征APE1和POLβ在 血管内皮生长因子G4启动子(目标1)。在指导阶段，应聘者也将利用 堪萨斯大学医学中心可用于专业发展的资源 并将通过结构化教学、指导、数据演示、 还有写作的机会。在项目的非指导阶段(R00)，技术技能 已获得的候选将用于阐明转录因子的招募模型 连接到血管内皮生长因子G4启动子序列(目的2)。此外，在R00阶段，候选人将延长 这些方法来询问误码率是如何在独特的G4底物上完成的，并阐明 修复和转录之间的耦合程度(目标3)。这些实验将提供 具有早期独立出版所需数据的候选人以及 R系列助学金。重要的是，在R00阶段，候选人将独立于 他们的导师专注于DNA修复和转录调控之间的相互作用 转移他们的工作，研究氧化DNA损伤作为转录的表观遗传学作用 通过DNA修复的调节器。

Project Summary/Abstract Environmental exposures and lifestyle choices can result in cellular oxidative stress, characterized by the generation of an abundance of reactive oxygen species (ROS). ROS wreak havoc on the structure of DNA bases, with guanine modification yielding the lesion 8-oxo-7,8- dihydroguanine (8oxoG) being particularly prevalent. If not repaired, 8oxoG is mutagenic, causing G to T transversion mutations that can initiate and promote human disease. Guanine-rich G- quadruplex (G4) forming sequences are enriched at promoter proximal regions of the genome, making these regions hot spots for 8oxoG lesions. The repair of 8oxoG by the base excision repair (BER) pathway on G4 promoter sequences (i.e., VEGF) can modulate transcription, however the molecular level interactions and mechanistic details of the repair activity within the G4 promoter context are not well understood. The overall goal of the proposed research is to characterize the molecular level interactions and coordination events coupling the repair of 8oxoG and gene enhancement at the VEGF promoter in response to oxidative stress. The experiments proposed to address this will be conducted in two phases. During the initial mentored K99 phase, X-ray crystallography, advanced nucleic acid kinetics, single-molecule fluorescence, and a human cell- based transcription assays will be utilized to characterize the activities of APE1 and Polβ on the VEGF G4 promoter (Aim 1). While in the mentored phase, the candidate will also take advantage of the resources available at University of Kansas Medical Center for professional development and will continuously apply these skills through structured teaching, mentoring, data presentation, and writing opportunities. During the non-mentored phase (R00) of the project, technical skills the candidate has gained will be used to elucidate a model for the recruitment of transcription factors to the VEGF G4 promoter sequence (Aim 2). Also, during the R00 phase the candidate will extend these approaches to interrogate how BER is completed on the unique G4 substrate and elucidate the extent of coupling between repair and transcription (Aim 3). These experiments will provide the candidate with the data required for an early independent publication and preliminary data for R-series grants. Importantly, during the R00 phase the candidate will develop independence from their mentor by focusing on the interplay between DNA repair and transcription regulation and shifting their work to study the epigenetic-like role of oxidative DNA lesions as transcription modulators through DNA repair.