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

项目总结/摘要 尽管真核生物的DNA通过称为核小体的重复单位包装成染色质， 通过活性氧（ROS）不断被破坏。8氧代鸟嘌呤（8 oxoG）是DNA的一种常见形式 由于鸟嘌呤氧化而造成的损伤。如果不修复，8 oxoG是诱变性的，导致G到T的颠换 这些突变可以引发和促进基因组不稳定性，最终导致人类疾病，如癌症。的 细胞对8 oxoG的主要防御是碱基切除修复（BER）途径。两种BER蛋白参与了 8 oxoG最初识别和去除是8 oxoGDNA糖基化酶1（OGG 1）和脱嘌呤/脱嘧啶 核酸内切酶1（APE 1）。OGG 1和APE 1必须发现、进入和修复复杂的基因组DNA损伤。 染色质结构，其中DNA被包装成核小体。核小体是一个重要的屏障 OGG 1和APE 1的活性，当损伤位于核小体附近时， 入口/出口部位。重要的是，已知进入/退出部位是高度动态的，并且经历自发和自发的变化。 核小体DNA的可逆解包和重包，从而为蛋白质提供DNA的通路 约束力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。尽管它 作为理解氧化DNA损伤如何在染色质内修复的关键， OGG 1和APE 1如何实现这一点仍然是未知。为此，本提案的总体目标是 揭示了OGG 1和APE 1如何在染色质环境中访问和处理DNA损伤。该提案 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， OGG 1和APE 1进入和处理DNA损伤。为了验证这一假设，有三个具体目标， 开发了整合强大和互补的生物物理技术，以提供广泛的洞察力， OGG 1和APE 1在染色质中的DNA损伤和修复。目的1将确定核小体DNA动力学如何 调节OGG 1和APE 1访问DNA损伤使用单分子荧光显微镜。目标2将 确定组蛋白PTM如何进一步调节OGG 1和APE 1的DNA损伤访问和处理， 单分子荧光显微术和DNA酶学。最后，目标3将阐明分子基础 使用冷冻电子显微镜观察OGG 1和APE 1与受损核小体的相互作用。完成 这些目标将提供一个全面的了解如何DNA损伤修复的背景下， 染色质，同时提供最先进的生物物理技术培训。这一创新举措将 在堪萨斯大学医学中心进行，由优秀的导师团队指导。在 除了研究部分外，该提案还纳入了一个强调职业和 专业发展。最终，本提案将提供必要的技能和专门知识， 申请人在DNA损伤修复和染色质的界面上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.