Repair of Oxidative Damage in G-Quadruplex Promoter DNA

G-四链体启动子 DNA 氧化损伤的修复

基本信息

批准号：
10226323
负责人：
Amy Michelle Whitaker
金额：
$ 10.62万
依托单位：
UNIVERSITY OF KANSAS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2022-03-05
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10226323
关键词：
Address Back Base Excision Repairs Beverages Binding Binding Sites Biochemical Biological Biological Assay C-terminal Cells Complement Consequentialism Consumption Coupled Coupling DNA DNA Damage DNA Repair DNA Sequence DNA Structure DNA lesion DNA ligase III Data Development Educational process of instructing Environmental Exposure Enzymatic Biochemistry Epigenetic Process Equilibrium Event Excision Exposure to Fluorescence Food Foundations G-Quartets Gene Activation Gene Expression Gene-Modified Generations Genes Genetic Transcription Genome Goals Grant Guanine Hot Spot Human Hypoxia Kansas Kinetics Lesion Life Style Medical center Mentors Modeling Modification Molecular Molecular Conformation N-terminal Nucleic Acids OGG1 gene Oxidation-Reduction Oxidative Stress Pathway interactions Phase Promoter Regions Proteins Publications Reaction Reactive Oxygen Species Research Research Personnel Resources Role Series Site Structure Technical Expertise Techniques Testing Time Training Transcriptional Activation Transcriptional Regulation Ultraviolet Rays United States National Institutes of Health Universities Vascular Endothelial Growth Factors Work Writing X-Ray Crystallography XRCC1 gene activating transcription factor base biophysical analysis experimental study human disease interdisciplinary approach oxidative DNA damage oxidative damage pollutant promoter reconstitution recruit repair enzyme repaired response single molecule single-molecule FRET skills transcription factor transversion mutation

项目摘要

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.

项目概要/摘要环境暴露和生活方式的选择会导致细胞氧化应激，其特点是产生大量的活性氧（ROS）。对 DNA 碱基结构造成严重破坏，鸟嘌呤修饰会产生损伤 8-oxo-7,8- 二氢鸟嘌呤 (8oxoG) 特别普遍，如果不进行修复，8oxoG 具有致突变性，导致。 G 到 T 颠换突变可以引发和促进富含鸟嘌呤的 G- 疾病。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8oxoG损伤的热点通过碱基切除修复来修复8oxoG。 G4 启动子序列（即 VEGF）上的 (BER) 通路可以调节转录，但是 G4启动子内修复活性的分子水平相互作用和机制细节所提出的研究的总体目标是描述背景。 8oxoG 和基因修复耦合的分子水平相互作用和协调事件实验提出，VEGF 启动子对氧化应激的反应增强。为了解决这个问题，将在最初的 K99 指导阶段进行 X 射线检查。晶体学、先进的核酸动力学、单分子荧光和人体细胞- 基于转录测定的方法将用于表征 APE1 和 Polβ 对 VEGF G4 启动子（目标 1）在指导阶段，候选人也将利用这一优势。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、数据演示不断应用这些技能，在项目的非指导阶段（R00），技术技能。候选人获得的成果将用于阐明转录因子招募的模型此外，在 R00 阶段，候选者将延伸至 VEGF G4 启动子序列。这些方法可探究 BER 如何在独特的 G4 基板上完成并阐明这些实验将提供修复和转录之间的耦合程度（目标 3）。候选人拥有早期独立出版物所需的数据和初步数据重要的是，在 R00 阶段，候选人将独立发展。他们的导师专注于 DNA 修复和转录调控之间的相互作用他们的工作转向研究氧化 DNA 损伤作为转录的表观遗传学作用通过 DNA 修复调节剂。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Transcriptional regulation via strand displacement DNA repair in G-quadruplexes.

通过 G-四链体中链置换 DNA 修复进行转录调控。

DOI：
发表时间：
2022
期刊：
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
影响因子：
0
作者：
Whitaker,Amy;Freudenthal,Bret
通讯作者：
Freudenthal,Bret

A two-residue nascent-strand steric gate controls synthesis of 2'-O-methyl- and 2'-O-(2-methoxyethyl)-RNA.

DOI：
10.1038/s41557-022-01050-8
发表时间：
2023-01
期刊：
Nature chemistry
影响因子：
21.8
作者：
Freund N;Taylor AI;Arangundy-Franklin S;Subramanian N;Peak-Chew SY;Whitaker AM;Freudenthal BD;Abramov M;Herdewijn P;Holliger P
通讯作者：
Holliger P

Processing oxidatively damaged bases at DNA strand breaks by APE1.