The role of cysteines in the response of 8-oxoguanine glycosylase (OGG1) to oxidative stress

项目摘要/摘要：暴露于环境毒素引起的氧化应激导致DNA 由DNA修复机制修复的损伤。8-氧代鸟嘌呤（8-oxoG）是最常见的 DNA损伤，其被8-氧代鸟嘌呤糖基化酶（OGG 1）识别并去除。OGG 1的功能是 重要的是防止DNA错误的传播和介导负责基因的转录， 氧化应激反应。细胞无法执行这些任务可能导致自身免疫性疾病， 神经退行性疾病、癌症和衰老。除了DNA氧化，半胱氨酸也可以被靶向 用于在氧化应激下的氧化修饰。OGG 1含有8个半胱氨酸残基， 在环境毒素的存在下进行修饰。本建议旨在阐明 OGG 1中的半胱氨酸在DNA修复和转录中对氧化应激的反应。生物化学表征 在具体目标1中提出了OGG 1半胱氨酸突变体的研究，以观察半胱氨酸在DNA结合中的功能， 酶活性、构象变化和蛋白质-蛋白质相互作用。这些研究将在 在K99指导阶段，然后在R 00阶段独立继续。第二个目标将 专注于体内细胞培养工作，以了解半胱氨酸在DNA修复和基因调控中的作用， 还鉴定由环境毒素处理引起的OGG 1修饰。在这方面拟议的工作 目标将在指导阶段启动，在指导阶段获得的技术专门知识 将在我独立工作期间受雇，以实现所提出的目标。这项工作将阐明作用 OGG 1修饰在疾病发生和进展中的作用，并将有助于预测OGG 1修饰的生物学效应。 毒素暴露这项工作也将为靶向药物设计提供基础。长期目标是 奖项是过渡到一个独立的学术研究计划，探索全面的分子 氧化应激下DNA损伤反应的机制以及DNA修复中的修饰 机器导致疾病。为了实现这个奖项的目标，我组建了一个导师团队， 环境科学、蛋白质组学、氧化还原生物化学、DNA糖基化酶、DNA修复和基因 转录。该团队还将为我提供职业发展方面的指导，以便过渡到 独立性和建立和运行一个成功的实验室。进一步的培训将从参加 在大学内外举办关于赠款撰写和职业发展的专题讲习班和课程 耶鲁大学参加和出席会议以及指导学生的额外机会， 博士后沿着继续准备手稿将是我的长期目标的补充， 建立独立的研究计划。这些工具对于获得技术培训和 职业发展，建立一个成功的研究计划，研究DNA的分子机制 氧化应激下的损伤反应及其在疾病中的作用。

Project Summary/Abstract: Oxidative stress caused from exposure to environmental toxins results in DNA damage that is repaired by the DNA repair machinery. 8-oxoguanine (8-oxoG) is the most commonly formed DNA lesion, which is recognized and removed by 8-oxoguanine glycosylase (OGG1). The function of OGG1 is important to prevent propagation of DNA error and to mediate transcription of genes responsible for the response to oxidative stress. The inability of cells to perform these tasks can lead to autoimmune and neurodegenerative diseases, cancer, and aging. In addition to DNA oxidation, cysteines can also be targeted for oxidative modification under oxidative stress. OGG1 contains 8 cysteine residues which are potentially targeted for modification in the presence of environmental toxins. This proposal seeks to elucidate the role of cysteine in OGG1 in DNA repair and transcription in response to oxidative stress. Biochemical characterization of OGG1 cysteine mutants is proposed in specific aim 1 to look at the function of cysteine in DNA binding, enzymatic activity, conformational changes, and in protein-protein interactions. These studies will be initiated during the K99 mentored phase and then continued independently during the R00 phase. The second aim will focus on in vivo cell culture work to understand the role of cysteine in DNA repair and in gene regulation and also to identify OGG1 modifications resulting from environmental toxin treatment. The proposed work in this aim will be initiated during the mentored phase, and the technical expertise gained during the mentored phase will be employed during my independent work to accomplish the proposed goals. This work will clarify the role of OGG1 modification in disease initiation and progression and will help in predicting the biological effect of toxin exposure. This work will also provide a foundation for targeted drug design. The long-term goal for this award is to transition into an independent academic research program to explore the comprehensive molecular mechanism of the DNA damage response under oxidative stress and how modifications in the DNA repair machinery lead to disease. To achieve the goals of this award, I assembled a team of mentors with expertise in environmental science, proteomics, redox biochemistry, DNA glycosylases, DNA repair, and gene transcription. This team will also provide me with mentoring in career development for transitioning into independence and establishing and running a successful lab. Further training will be acquired from attending special topic workshops and courses on grant writing and career development offered both at and outside of Yale University. Additional opportunities to attend and present at conferences and to mentor students and postdocs along with continued preparation of manuscripts will be complementary for my long-term goal of establishing an independent research program. These tools will be essential to gain technical training and for career development to establish a successful research program to study the molecular mechanism of the DNA damage response under oxidative stress and its role in disease.