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

项目摘要/摘要 尽管真核DNA通过被称为核小体的重复单位包装成染色质，但它 通过活性氧物种(ROS)不断受到损害。8oxo-guanine(8oxoG)是DNA的一种常见形式 鸟嘌呤氧化造成的损害。如果不修复，8oxoG会发生突变，导致G到T的颠换 突变可以引发和促进基因组的不稳定，并最终导致人类疾病，如癌症。这个 细胞对8oxoG的主要防御是碱基切除修复(BER)途径。两种误码率蛋白参与 8oxoG的最初识别和去除是8oxoG DNA糖基酶1(OGG1)和脱嘌呤/脱嘧啶 核酸内切酶1(APE1)。OGG1和APE1必须在复合体中发现、访问和修复基因组DNA损伤 染色质结构，DNA被包装成核小体。核小体是一个重要的障碍 对OGG1和APE1活性的影响，当损伤位于核小体附近时，这种活性减轻 出入境地点。重要的是，出入境地点是众所周知的高度动态的，并经历自发和 核小体DNA的可逆解包装和重新包装，从而为蛋白质提供了获得DNA的途径 有约束力的。核小体动力学通过对核小体的翻译后修饰(PTM)进一步调节 核小体，允许细胞在不同的细胞条件下微调对DNA的访问。尽管如此 对于理解染色质内氧化DNA损伤是如何修复的至关重要，对 OGG1和APE1如何做到这一点仍然令人费解。为此，这项提案的首要目标是 揭示OGG1和APE1如何在染色质环境中访问和处理DNA损伤。该提案是 基于核小体DNA动力学和组蛋白PTM是关键调控决定因素的假设 OGG1和APE1访问和处理DNA损伤。为了验证这一假设，有三个具体目标 集成了强大和互补的生物物理技术以提供广泛的洞察力 OGG1和APE1对染色质DNA的损伤与修复目标1将确定核小体DNA动力学如何 利用单分子荧光显微镜调控OGG1和APE1对DNA损伤的通路。目标2将 确定组蛋白PTMS如何利用OGG1和APE1进一步调节DNA损伤通路和处理 单分子荧光显微镜和DNA酶学。最后，目标3将阐明分子基础。 用冷冻电子显微镜观察OGG1和APE1与受损核小体的相互作用。完成 这些目标将提供对DNA损伤如何在以下背景下修复的全面理解 染色质，同时提供最先进的生物物理技术培训。这项创新的建议将是 在堪萨斯大学医学中心一个优秀的导师团队的指导下进行。在……里面 除研究部分外，该提案还纳入了一项培训计划，强调职业和 职业发展。最终，这项提议将提供必要的技能和专业知识 申请者在DNA损伤修复和染色质之间建立一个富有成效的独立研究小组。

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.