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-氧气 - 瓜氨酸是最常见和高度诱变的氧化病变之一，因为它误解了在DNA复制过程中与腺嘌呤。碱性切除修复主要消除了氧化的碱病变（BER）途径。 BER与DNA损伤响应（DDR）紧密协调以保持基因组稳定性和细胞存活。尽管BER和DDR分别进行了很好的研究，但两者的协调过程不了解。异三聚合物9-1-1（RAD9-RAD1-HUS1）检查点夹在中扮演双重角色 DDR和DNA修复过程的激活。我们已经确定了BER和DDR之间的独特互动蛋白质表现出9-1-1对BER的新颖和关键贡献。我们假设9-1-1提供了协调ber过程以避免有毒中间体积累的平台。这个项目的目标是定义介导初始的9-1-1和两种酶之间的生化和功能关系伯的步骤。 MYH/MUTYH DNA糖基酶消除了与8-氧化的腺苷掺入的腺丝鸟嘌呤在BER的第一步中预防基因突变。 ape1核酸内切酶随后在在BER的第二步中，无卑鄙的站点。该协调的研究很重要，因为这些蛋白质是过程中的主要参与者是端粒维持和预防人类疾病。已经表明人MYH基因的突变与大肠癌有关，而APE1和9-1-1对于细胞活力和发育。提出了以下三个特定目标。（1）我们将定义功能 DNA修复复合物由MYH，APE1和9-1-1组成。我们将检查该维修的形成是否复合物对于通过中断蛋白质 - 蛋白质相互作用。（2）我们将测试一个模型，即每个9-1-1的亚基在BER和DDR中都起着独特的作用。我们建议RAD9稳定在DNA上的BER机械，HUS1促进了平稳的传递从MYH到APE1的有毒中间。（3）我们将采用新型的诱导ROS系统来限制DNA 损坏单个基因组位置和端粒。我们将使用这些系统比较BER率和两个位置的BER因子关联顺序。我们将检查BER因子的相互依赖性使用敲低和敲除方法与DNA损伤部位相关联。这些研究将揭示确切的BER综合体是如何在病变部位组装好的，而BER为何对保持端粒完整性。从这些研究中获得的见解将大大推动我们的了解BER和DDR在癌变，癌症治疗和衰老中的作用。因为DNA 维修和9-1-1介导的信号传导与癌症的发展和治疗有关，中断这些协调的过程可以为开发抗癌药物提供创新的策略。