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.

项目摘要/摘要环境暴露和生活方式的选择会导致细胞氧化物应激，其特征是产生丰富的活性氧（ROS）。罗斯屈服 DNA碱基结构的破坏，鸟嘌呤的修饰产生了8-oxo-7,8-的病变二氢甘氨酸（8oxog）特别普遍。如果不修理，8oxog是诱变的，导致 g转移突变，可以引发和促进人类疾病。鸟嘌呤丰富的g- 四链体（G4）形成序列富含基因组的启动子代理区域，使这些区域用于8OXOG病变。基本惊喜维修对8oxog的维修（BER）G4启动子序列（即VEGF）的途径可以调节转录，但是 G4启动子内修复活性的分子水平相互作用和机械细节上下文不太了解。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，结合了8oxog和基因的修复响应氧化应激的VEGF启动子的增强。提出了实验为此解决这将分为两个阶段。在最初修补的K99阶段，X射线晶体学，晚期核酸动力学，单分子荧光和人类细胞基于基于的转录测定将用于表征APE1和POLβ在 VEGF G4启动子（AIM 1）。在修订阶段，候选人也将利用堪萨斯大学医学中心专业发展的资源并将继续通过结构化教学，心理，数据表现来运用这些技能，和写作机会。在项目的未批准阶段（R00）期间，技术技能获得的候选人将用于阐明募集转录因子的模型到VEGF G4启动子序列（AIM 2）。同样，在R00阶段，候选人将延长这些方法来询问BER如何在唯一的G4底物上完成并阐明维修和转录之间的耦合程度（AIM 3）。这些实验将提供候选人拥有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R00阶段，候选人将发展独立于通过关注DNA修复与转录调节和转录调节之间的相互作用以及转移他们的工作以研究氧化DNA病变的表观遗传样作用通过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.