Transcription-coupled repair of Oxidative DNA damage in vivo

体内氧化 DNA 损伤的转录偶联修复

基本信息

批准号：
8061606
负责人：
Justin Courcelle
金额：
$ 25.21万
依托单位：
PORTLAND STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-01 至 2013-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8061606
关键词：
Address Aging Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Animal Model Antioxidants Apoptosis Apoptotic Arabinose Biological Assay Cell Culture Techniques Cell Death Cell Line Cell physiology Cells Cockayne Syndrome Coupled Coupling DNA DNA Damage DNA Repair DNA glycosylase DNA lesion DNA-Directed RNA Polymerase Development Disease Escherichia coli Essential Genes Fanconi&apos s Anemia Friedreich Ataxia Genes Genetic Transcription Genome Health Hereditary Disease Human Inherited Kinetics Lactose Lead Lesion Malignant Neoplasms Malignant neoplasm of ovary Mammalian Cell Measures Mutation Necrosis Nervous System Physiology Neurologic Nucleotide Excision Repair Operon Oxygen Paper Parkinson Disease Patients Pharmaceutical Preparations Play Process Proteins RNA chemical synthesis Reactive Oxygen Species Recovery Recruitment Activity Relative (related person)Reporting Research Role Series Signal Transduction Substrate Specificity Testing Transcription-Coupled Repair UV induced chemotherapy human disease in vivo malignant breast neoplasm mutant novel novel therapeutic intervention oxidative DNA damage oxidative damage prevent repaired thymine glycol

项目摘要

DESCRIPTION (provided by applicant): DNA damage that blocks transcription can prevent the expression of essential genes, leading to mutations, apoptosis, or necrotic cell death. Transcription-coupled repair is a cellular process by which some forms of DNA damage are repaired more rapidly from transcribed strands of active genes than from nontranscribed strands or the overall genome. Cockayne syndrome patients are characterized by developmental and neurological deficiencies and are specifically defective in the process transcription-coupled repair. It has been widely speculated that the transcription-coupled repair of oxidative-DNA lesions, in particular, may be an underlying cause of the underlying developmental and neurological deficiencies in Cockayne's syndrome, and may be involved in other diseases that involve the progressive loss of neurological function, such as Parkinsons and Alzheimer's disease. However, the rapid kinetics of oxidative repair relative to transcription, and the apoptotic cascade induced by reactive oxygen and stalled transcription machinery have made it technically difficult to address this hypothesis in mammalian cells, despite intense efforts. We therefore, propose to test this hypothesis directly in the model organism of E.coli, where the process of transcription-coupled repair and oxidative DNA repair are highly conserved. We show that the low complexity genome, well-characterized transcriptional operons, and use of purified DNA glycosylases and isogenic mutants allow us to overcome the obstacles arising in human cell cultures to detect and definitively answer this important question. We hypothesize that specific oxidative DNA lesions are repaired in a transcription-coupled manner in vivo. We further hypothesize that lesions that block RNA polymerase will be subject to transcription-coupled repair, whereas nonblocking lesions will not, and that the process will depend on a number of gene products including, a coupling factor- Mfd, nucleotide excision repair, and specific DNA glycosylases. To test these hypotheses, we will 1) use purified DNA glycosylases with known substrate specificities to measure the repair kinetics of different oxidative DNA lesions in vivo; 2) examine the repair rates of different classes of oxidative damage, 8-oxoguanine, thymine glycol, and others, to identify which classes of oxidative lesions are repaired in a transcription-coupled manner; 3) measure the repair rate of oxidative lesions and recovery of RNA synthesis in isogenic mutants that lack nucleotide excision repair, oxidative DNA glycosylases, or Mfd. PUBLIC HEALTH RELEVANCE: The results from this project will enhance our understanding of the roles of transcription and transcription-coupled repair in processing oxidative DNA damage that have been implicated in human disease. Reactive oxygen species are directly or indirectly associated with a range of human hereditary diseases ranging from Parkinsons and Alzheimers, to amyotrophic lateral sclerosis and Friedreich's ataxia, to Fanconi anemia and Cockayne syndrome. In addition, there is increasing evidence to suggest reactive oxygen species play a significant role in the spontaneous cancers and aging. Since both oxidative DNA damage and transcription arrest generate strong signals for apoptosis, the research may lead to novel modes of chemotherapy, involving selective inhibition of transcription-coupled repair in target cells combined with administration of transcription-blocking drugs or antioxidants.

描述（由申请人提供）：阻碍转录的 DNA 损伤可以阻止必需基因的表达，导致突变、细胞凋亡或坏死性细胞死亡。转录偶联修复是一种细胞过程，通过该过程，活性基因的转录链比非转录链或整个基因组修复某些形式的 DNA 损伤的速度更快。科凯恩综合征患者的特征是发育和神经缺陷，并且在转录偶联修复过程中特别有缺陷。人们普遍推测氧化DNA损伤的转录偶联修复可能是科凯恩综合征中潜在的发育和神经缺陷的根本原因，并且可能涉及其他涉及神经功能进行性丧失的疾病。功能，例如帕金森病和阿尔茨海默病。然而，尽管付出了巨大的努力，但氧化修复相对于转录的快速动力学，以及活性氧诱导的细胞凋亡级联和停滞的转录机制，使得在哺乳动物细胞中在技术上难以解决这一假设。因此，我们建议直接在大肠杆菌模型生物中测试这一假设，其中转录偶联修复和氧化 DNA 修复过程高度保守。我们证明，低复杂性的基因组、特征明确的转录操纵子以及纯化的 DNA 糖基酶和同基因突变体的使用使我们能够克服人类细胞培养中出现的障碍，以检测并明确回答这一重要问题。我们假设特定的氧化 DNA 损伤在体内以转录耦合方式修复。我们进一步假设，阻断 RNA 聚合酶的病变将受到转录偶联修复，而非阻断性病变则不会，并且该过程将取决于许多基因产物，包括偶联因子 Mfd、核苷酸切除修复和特异性基因产物。 DNA糖基化酶。为了检验这些假设，我们将1）使用具有已知底物特异性的纯化DNA糖基化酶来测量体内不同氧化DNA损伤的修复动力学； 2）检查不同类别的氧化损伤、8-氧代鸟嘌呤、胸腺嘧啶乙二醇等的修复率，以确定哪些类别的氧化损伤以转录偶联方式修复； 3) 测量缺乏核苷酸切除修复、氧化DNA糖基化酶或Mfd的等基因突变体中氧化损伤的修复率和RNA合成的恢复率。公共健康相关性：该项目的结果将增强我们对转录和转录偶联修复在处理与人类疾病有关的氧化 DNA 损伤中的作用的理解。活性氧与一系列人类遗传性疾病直接或间接相关，包括帕金森病和阿尔茨海默病、肌萎缩侧索硬化症和弗里德赖希共济失调、范可尼贫血和科凯恩综合征。此外，越来越多的证据表明活性氧在自发性癌症和衰老中发挥着重要作用。由于DNA氧化损伤和转录抑制都会产生强烈的细胞凋亡信号，因此这项研究可能会带来新的化疗模式，包括选择性抑制靶细胞中的转录偶联修复，并结合施用转录阻断药物或抗氧化剂。