Repair of Oxidative Damage in G-Quadruplex Promoter DNA

项目总结/摘要 环境暴露和生活方式的选择会导致细胞氧化应激， 其特征在于产生大量活性氧（ROS）。ROS wreak 破坏DNA碱基的结构，鸟嘌呤修饰产生损伤8-氧代-7，8- 二氢鸟嘌呤（8 oxoG）尤其普遍。如果不修复，8 oxoG是诱变性的， G到T颠换突变可以引发和促进人类疾病。富鸟嘌呤G- 四链体（G4）形成序列在基因组的启动子近端区域富集， 使这些区域成为8 oxoG损伤的热点。通过碱基切除修复对8 oxoG的修复 (BER)G4启动子序列上的途径（即，VEGF）可以调节转录，然而， G4启动子内修复活性的分子水平相互作用和机制细节 上下文没有很好地理解。拟议研究的总体目标是表征 分子水平的相互作用和协调事件耦合8 oxoG和基因的修复 VEGF启动子对氧化应激的反应增强。提出的实验 将分两个阶段进行。在最初的指导K99阶段，X射线 晶体学，先进的核酸动力学，单分子荧光，和人类细胞- 将利用基于转录的测定来表征APE 1和Polβ在 VEGF G4启动子（Aim 1）。在辅导阶段，候选人还将利用 堪萨斯大学医学中心的专业发展资源 并将通过结构化教学，指导，数据展示， 写作的机会。在项目的非指导阶段（R 00）， 候选人已经获得将用于阐明转录因子的招募模型 VEGF G4启动子序列（Aim 2）。此外，在R 00阶段，候选人将扩展 这些方法来询问BER是如何在独特的G4基板上完成的，并阐明 修复和转录之间的偶联程度（目的3）。这些实验将提供 候选人与早期独立出版物所需的数据和初步数据， R系列赠款。重要的是，在R 00阶段，候选人将独立于 他们的导师通过专注于DNA修复和转录调控之间的相互作用， 将他们的工作转移到研究DNA氧化损伤在转录过程中的表观遗传作用， 通过DNA修复调节剂。

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.