Base Excision Repair: Mechanisms of DNA Damage Access and Repair in Chromatin

碱基切除修复：染色质中 DNA 损伤接近和修复的机制

• 批准号: 10154528
• 负责人: Tyler Weaver
• 金额: $ 6.6万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-16 至 2022-12-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10154528
• 关键词: 8-hydroxyguanosine; Address; Base Excision Repairs; Binding; Cells; Chromatin; Chromatin Structure; Code; Complex; Cryoelectron Microscopy; DNA; DNA Damage; DNA Repair; DNA glycosylase; DNA-Binding Proteins; Development; Environment; Enzymatic Biochemistry; Enzyme Kinetics; Eukaryota; Excision; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Genome Scan; Genomic DNA; Genomic Instability; Goals; Grant; Guanine; Histones; Kansas; Kinetics; Lead; Left; Lesion; Link; Malignant Neoplasms; Medical center; Mentorship; Molecular; Monitor; Nucleosomes; Oxidative Stress; Pathway interactions; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Reactive Oxygen Species; Regulation; Research; Site; Structure; Techniques; Testing; Time; Training; Universities; base; biophysical techniques; career; endonuclease; experimental study; histone modification; human disease; innovation; insight; kinetic model; oxidation; oxidative DNA damage; repair function; repaired; single molecule; skills; sugar; transversion mutation

Project Summary/Abstract Despite the packaging of eukaryotic DNA into chromatin through repeating units known as the nucleosomes, it is constantly damaged via reactive oxygen species (ROS). 8oxo-guanine (8oxoG) is a common form of DNA damage resulting from the oxidation of guanine. If not repaired, 8oxoG is mutagenic, causing G to T transversion mutations that can initiate and promote genomic instability and ultimately human disease, such as cancer. The cells primary defense against 8oxoG is the base excision repair (BER) pathway. Two BER proteins involved in the initial recognition and removal of 8oxoG are 8oxoG DNA glycosylase 1 (OGG1) and apurinic/apyrimidinic endonuclease 1 (APE1). OGG1 and APE1 must find, access, and repair genomic DNA damage in complex chromatin structures, where the DNA is packaged into nucleosomes. Nucleosomes present a significant barrier to the activities of OGG1 and APE1, which is alleviated when the damage is positioned near the nucleosome entry/exit site. Importantly, the entry/exit site is known to be highly dynamic and undergoes spontaneous and reversible unwrapping and rewrapping of the nucleosomal DNA, thus providing access to the DNA for protein binding. Nucleosome dynamics are further regulated through post-translational modifications (PTMs) to the nucleosome, which allow the cell to fine-tune access to the DNA under different cellular conditions. Despite it being critical to understanding how oxidative DNA damage is repaired within chromatin, mechanistic insight into how OGG1 and APE1 accomplish this remains elusive. To this end, the overarching goal of this proposal is to reveal how OGG1 and APE1 access and process DNA damage in a chromatin environment. The proposal is based on the hypothesize that nucleosomal DNA dynamics and histone PTMs are key regulatory determinants for OGG1 and APE1 to access and process DNA damage. To test this hypothesis, three specific aims have been developed that integrate powerful and complementary biophysical techniques to provide extensive insight into DNA damage and repair in chromatin by OGG1 and APE1. Aim 1 will determine how nucleosomal DNA dynamics regulate OGG1 and APE1 access to DNA damage using single-molecule fluorescence microscopy. Aim 2 will determine how histone PTMs further regulate DNA damage access and processing by OGG1 and APE1 using single-molecule fluorescence microscopy and DNA enzymology. Finally, Aim 3 will elucidate the molecular basis for OGG1 and APE1 interactions with damaged nucleosomes using cryo-electron microscopy. Completion of these aims will provide a comprehensive understanding of how DNA damage is repaired in the context of chromatin, while providing training in state-of-the-art biophysical techniques. This innovative proposal will be carried out at the University of Kansas Medical Center under the guidance of an excellent mentorship team. In addition to the research component, the proposal incorporates a training plan that emphasizes career and professional development. Ultimately, this proposal will provide the skills and expertise necessary for the applicant to build a productive independent research group at the interface of DNA damage repair and chromatin.

项目总结/文摘