R15 AREA: Replication in the Presence of Oxidative DNA damage

R15 区域：存在氧化 DNA 损伤时的复制

• 批准号: 8290917
• 负责人: Justin Courcelle
• 金额: $ 43.65万
• 依托单位: PORTLAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8290917
• 关键词: Address; Aging-Related Process; Amyotrophic Lateral Sclerosis; Animal Model; Antioxidants; Apoptosis; Biochemical; Biological Assay; Cell Survival; Cell division; Cell physiology; Cells; Cellular Assay; Chemicals; Chromosomes; Cockayne Syndrome; Coupled; DNA Repair; DNA glycosylase; Data; Development; Disease; Enzymes; Escherichia coli; Event; Fanconi' s Anemia; Friedreich Ataxia; Genes; Genetic Transcription; Genome; Hereditary Disease; Human; In Vitro; Individual; Lead; Lesion; Liquid Chromatography; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Measures; Monitor; Mutation; Nervous System Physiology; Neurologic; Oxidative Stress; Parkinson Disease; Pathway interactions; Play; Polymerase; Process; Reactive Oxygen Species; Recovery; Research; Research Personnel; Role; Signal Transduction; Substrate Specificity; Syndrome; Testing; UV induced; aged; base; human disease; in vivo; inhibitor/antagonist; liquid chromatography mass spectrometry; mutant; normal aging; novel; novel therapeutic intervention; oxidative DNA damage; oxidative damage; repair enzyme; repaired; response; sensor; Address; Aging-Related Process; Amyotrophic Lateral Sclerosis; Animal Model; Antioxidants; Apoptosis; Biochemical; Biological Assay; Cell Survival; Cell division; Cell physiology; Cells; Cellular Assay; Chemicals; Chromosomes; Cockayne Syndrome; Coupled; DNA Repair; DNA glycosylase; Data; Development; Disease; Enzymes; Escherichia coli; Event; Fanconi' s Anemia; Friedreich Ataxia; Genes; Genetic Transcription; Genome; Hereditary Disease; Human; In Vitro; Individual; Lead; Lesion; Liquid Chromatography; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Measures; Monitor; Mutation; Nervous System Physiology; Neurologic; Oxidative Stress; Parkinson Disease; Pathway interactions; Play; Polymerase; Process; Reactive Oxygen Species; Recovery; Research; Research Personnel; Role; Signal Transduction; Substrate Specificity; Syndrome; Testing; UV induced; aged; base; human disease; in vivo; inhibitor/antagonist; liquid chromatography mass spectrometry; mutant; normal aging; novel; novel therapeutic intervention; oxidative DNA damage; oxidative damage; repair enzyme; repaired; response; sensor

DESCRIPTION (provided by applicant): Project Summary Oxidative DNA damage is associated with a range of human disease states involving the progressive loss of developmental and neurological functions, including Cockaynes syndrome, Parkinsons, and Alzhiemers disease. It is by far, the most common form of damage encountered by cells, and it is widely speculated that disease is the result of a gradual accumulation of damage and mutations that eventually compromises cellular function or viability in repair compromised or naturally aged individuals. Yet despite these associations, the cellular mechanism by which oxidative lesions are processed during replication in vivo remains largely uncharacterized. In part, this is because cells devote a suite of enzymes to the repair and tolerance of oxidative damage that appear redundant in biochemical assays and in part because there is a lack of cellular assays that make it challenging to address in mammalian cells The results of this proposal will address these questions directly using the model organism of E.coli, where both replication and oxidative DNA repair are highly conserved. We have established unique cellular assays in E. coli to monitor replication fork processing, and our ability to rapidly purify glycosylases and generate mutants will allow us to directly and definitively determine the how oxidative lesions are processed in vivo. We describe three aims that will be accomplished. 1) Using a unique cellular assay to monitor the repair of lesion in vivo we will identify the biologically relevant oxidative DNA glycosylases that are responsible for the global repair of the genome and subseuqnetly identify the substrate lesions using LC/MS/MS and GC/MS. 2) We will identify the genes and mechanism by which a novel global sensor of oxidative stress transiently shuts downs replication and transcription in response to oxidative stress. 3) We will determine the contribution that repair and translesion synthesis have in processing oxidative lesions encountered during replication and identify the intermediates that arise during the recovery of replication in the presence of these lesions. The results of these studies will allow researchers to determine whether specific oxidative repair deficiencies or impaired processing events lead to human disease states and may suggest novel therapeutic approaches targeting either these replication or repair pathways. PUBLIC HEALTH RELEVANCE: Significance: The results from this project will enhance our understanding of the specific roles that oxidative DNA damage has in causing human disease. Reactive oxygen species are directly or indirectly associated with a range of human hereditary diseases, including Parkinsons, Alzhiemers, amyotrophic lateral sclerosis, Friedreich's ataxia, Fanconi anemia, and Cockayne syndrome. In addition, there is increasing evidence to suggest that reactive oxygen species play a significant role in both spontaneous cancers and the normal aging process. Identifying the cellular role for oxidative repair enzymes and how lesions are processed during replication will allow researchers to examine whether specific repair deficiencies are causative of these human disease states. Furthermore, since oxidative DNA damage generates strong signals for apoptosis, the research may lead to novel modes of chemotherapeutics, involving selective inhibition of repair enzymes identified in this study combined with administration of replicational inhibitors or antioxidants.

描述（由申请人提供）： 项目摘要氧化DNA损伤与一系列人类疾病状态有关，涉及发育和神经功能的逐渐丧失，包括Cockaynes综合征，帕金森氏病和阿尔茨希默斯病。到目前为止，这是细胞遇到的最常见损害形式，并且广泛推测疾病是损害和突变逐渐积累的结果，最终损害了细胞功能或修复受损或自然老年人的细胞功能或生存能力。然而，尽管存在这些关联，但在体内复制过程中处理氧化病变的细胞机制仍然很大程度上没有表征。部分原因是，这是因为细胞将一组酶用于维修和容忍氧化损伤，这在生化测定中看起来多余，部分原因是缺乏细胞测定，这使得在哺乳动物细胞中解决该提案的结果具有挑战性。我们已经在大肠杆菌中建立了独特的细胞测定，以监测复制叉的加工，并且我们快速纯化糖基酶并产生突变体的能力将使我们能够直接，确定地确定在体内处理氧化性病变的方式。 我们描述了将实现的三个目标。 1）使用独特的细胞测定法监测体内病变的修复，我们将确定负责使用LC/MS/MS/MS/MS/MS/MS/MS和GC/MS的生物学上相关的氧化DNA糖基化酶，这些氧化性DNA糖基化酶均可识别基因组全球修复的底物病变。 2）我们将确定新型的氧化应激全球传感器瞬时对氧化应激的复制和转录的基因和机制。 3）我们将确定在复制过程中遇到的氧化病变中的修复和跨性质合成具有的贡献，并确定在存在这些病变的情况下复制过程中复制过程中出现的中间体。 这些研究的结果将使研究人员能够确定特定的氧化修复缺陷或加工事件受损是否导致人类疾病状态，并可能提出针对这些复制或修复途径的新型治疗方法。 公共卫生相关性： 意义：该项目的结果将增强我们对氧化DNA损害引起人类疾病的特定作用的理解。活性氧直接或间接与包括帕金森氏症，阿尔茨希默斯，肌萎缩性侧索硬化症，弗里德里希的共济失调，芬科尼尼贫血和cockayne综合征在内的一系列人类遗传疾病有关。此外，越来越多的证据表明，活性氧在自发癌和正常衰老过程中都起着重要作用。 确定氧化修复酶的细胞作用以及在复制过程中如何处理病变将使研究人员能够检查特定的修复缺陷是否是这些人类疾病状态的原因。此外，由于氧化DNA损伤会产生强烈的凋亡信号，因此该研究可能导致新型的化学疗法模式，涉及对本研究中鉴定的修复酶的选择性抑制，并结合了复制抑制剂或抗氧化剂的给药。