The role of the checkpoint clamp in DNA repair

检查点钳在DNA修复中的作用

基本信息

批准号：
9894102
负责人：
A-Lien L Lu-Chang
金额：
$ 9.3万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-03-01 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9894102
关键词：
8-hydroxyguanosine Adenine Affinity Aging Antineoplastic Agents Apoptosis Base Excision Repairs Binding Biochemical Biological Biological Process C-terminal Cell Cycle Cell Cycle Arrest Cell Survival Cells Cellular Assay Charge Clinical Closure by clamp Colorectal Cancer Complex Couples DNA DNA Binding DNA Damage DNA Repair DNA Repair Enzymes DNA Repair Pathway DNA biosynthesis DNA glycosylase DNA lesion DNA-(apurinic or apyrimidinic site) lyase Data Dependence Development Disease Drug resistance Enzymes Exposure to Fission Yeast Frequencies Gene Mutation Genes Genome Genome Stability Genomics Goals Guanine Human Interruption Kinetics Knock-out Lead Lesion Location Maintenance Malignant Neoplasms Measures Mediating Modeling Monitor Mutate Mutation Organism Oxidative Stress Oxides Pathway interactions Peptides Phosphorylation Phosphotransferases Photosensitivity Play Primary Lesion Process Proteins RAD9A gene Radiation Reactive Oxygen Species Repair Complex Role Signal Transduction Site System Tail Telomere Maintenance Testing base cancer therapy carcinogenesis colon cancer patients disorder prevention endonuclease genome integrity human disease in vivo innovation insight knock-down mutant neoplastic cell novel prevent protein protein interaction radiation resistance recruit repaired response sensor telomere tumor

项目摘要

Exposure to reactive oxygen species (ROS) and radiation leads to DNA damage that compromise genomic integrity. 8-oxo-guanine is one of the most frequent and highly mutagenic oxidative lesions because it mispairs with adenine during DNA replication. Oxidized base lesions are primarily eliminated by the base excision repair (BER) pathway. BER is tightly coordinated with DNA damage response (DDR) in order to maintain genomic stability and cell survival. Although BER and DDR have been well studied separately, the coordination of both processes is not understood. The heterotrimeric 9-1-1 (Rad9-Rad1-Hus1) checkpoint clamp plays dual roles in activation of DDR and DNA repair processes. We have identified unique interactions among BER and DDR proteins, demonstrating a novel and critical contribution of 9-1-1 to BER. We hypothesize that 9-1-1 provides a platform to coordinate BER processes to avoid the accumulation of toxic intermediates. The goal of this project is to define the biochemical and functional relationships between 9-1-1 and two enzymes that mediate initial steps of BER. The MYH/MUTYH DNA glycosylase excises misincorporated adenines paired with 8-oxo- guanine to prevent gene mutation in the first step of BER. APE1 endonuclease subsequently nicks DNA at abasic sites in the second step of BER. Studies of this coordination are important because these proteins are key players in processes as telomere maintenance and human disease prevention. It has been shown that mutations in human MYH gene are associated with colorectal cancer while APE1 and 9-1-1 are essential for cell viability and development. The following three specific aims are proposed. (1) We will define a functional DNA repair complex consisting of MYH, APE1, and 9-1-1. We will examine whether the formation of this repair complex is critical for their biological functions in maintaining genomic stability by interrupting the protein- protein interactions. (2) We will test a model that each subunit of 9-1-1 plays a distinct role in BER and DDR. We propose that Rad9 stabilizes BER machinery on DNA and that Hus1 promotes the smooth transfer of the toxic intermediate from MYH to APE1. (3) We will employ novel inducible ROS systems to confine DNA damage to a single genomic location and telomeres. We will use these systems to compare the BER rates and the order of BER factor association at both locations. We will examine the mutual dependence of BER factor association with sites of DNA damage using knockdown and knockout approaches. These studies will reveal exactly how the BER complex is assembled at lesion sites on fine scale and why BER is important for maintaining telomere integrity. The insights gained from these studies will significantly advance our understanding of the roles of BER and DDR in carcinogenesis, cancer treatment, and aging. Because DNA repair and 9-1-1 mediated signaling are associated with cancer development and treatment, interrupting these coordinated processes could provide an innovative strategy for the development of anticancer drugs.

暴露于活性氧 (ROS) 和辐射会导致 DNA 损伤，从而损害基因组正直。 8-氧代鸟嘌呤是最常见且高度致突变的氧化损伤之一，因为它会错配 DNA复制过程中与腺嘌呤结合。氧化的碱基病变主要通过碱基切除修复来消除（BER）路径。 BER 与 DNA 损伤反应 (DDR) 紧密协调，以维持基因组稳定性和细胞存活。尽管 BER 和 DDR 已经分别得到了很好的研究，但两者的协调流程不被理解。异三聚体 9-1-1 (Rad9-Rad1-Hus1) 检查点钳在 DDR 和 DNA 修复过程的激活。我们已经确定了 BER 和 DDR 之间独特的相互作用蛋白质，展示了 9-1-1 对 BER 的新颖且关键的贡献。我们假设 9-1-1 提供了平台协调 BER 流程以避免有毒中间体的积累。该项目的目标的目的是定义 9-1-1 和介导初始反应的两种酶之间的生化和功能关系 BER 的步长。 MYH/MUTYH DNA 糖基化酶切除与 8-oxo- 配对的错误掺入的腺嘌呤鸟嘌呤在 BER 的第一步中防止基因突变。 APE1 核酸内切酶随后在以下位置切割 DNA BER 第二步中的 abasic 站点。对这种协调的研究很重要，因为这些蛋白质端粒维护和人类疾病预防等过程中的关键参与者。事实证明人类 MYH 基因突变与结直肠癌相关，而 APE1 和 9-1-1 对于结直肠癌至关重要细胞活力和发育。提出以下三个具体目标。 (1) 我们定义一个泛函 DNA 修复复合物由 MYH、APE1 和 9-1-1 组成。我们会检查这个修复是否形成复合物通过中断蛋白质来维持基因组稳定性，对其生物学功能至关重要。蛋白质相互作用。 (2) 我们将测试一个模型，9-1-1 的每个子单元在 BER 和 DDR 中起着不同的作用。我们建议 Rad9 稳定 DNA 上的 BER 机制，而 Hus1 促进 DNA 上的 BER 机制的顺利转移。从 MYH 到 APE1 的有毒中间体。 (3)我们将采用新型诱导ROS系统来限制DNA 对单个基因组位置和端粒的损伤。我们将使用这些系统来比较 BER 率和两个位置的 BER 因子关联的顺序。我们将检查 BER 因子的相互依赖性使用击倒和敲除方法与 DNA 损伤位点关联。这些研究将揭示 BER 复合体到底是如何在病变部位进行精细组装的，以及为什么 BER 对维持端粒完整性。从这些研究中获得的见解将极大地推进我们的研究了解 BER 和 DDR 在致癌、癌症治疗和衰老中的作用。因为DNA 修复和 9-1-1 介导的信号传导与癌症的发展和治疗相关，中断这些协调的过程可以为抗癌药物的开发提供创新策略。