Repair of Oxidative Damage in G-Quadruplex Promoter DNA

G-四链体启动子 DNA 氧化损伤的修复

• 批准号: 10561776
• 负责人: Amy Michelle Whitaker
• 金额: $ 24.9万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-06 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10561776
• 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

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.

项目总结/文摘