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

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 氧化性 DNA 损伤与一系列涉及发育和神经功能逐渐丧失的人类疾病状态有关，包括科凯恩综合征、帕金森病和阿尔茨海默病。这是迄今为止细胞遇到的最常见的损伤形式，人们普遍推测疾病是损伤和突变逐渐积累的结果，最终损害修复受损或自然衰老个体的细胞功能或活力。然而，尽管存在这些关联，在体内复制过程中处理氧化损伤的细胞机制在很大程度上仍然未知。部分原因是细胞使用一套酶来修复和耐受氧化损伤，这些酶在生化检测中显得多余，部分原因是缺乏细胞检测，这使得在哺乳动物细胞中解决这一问题具有挑战性。该提案将直接使用大肠杆菌模型生物来解决这些问题，其中复制和氧化 DNA 修复都是高度保守的。我们在大肠杆菌中建立了独特的细胞测定法来监测复制叉加工，并且我们快速纯化糖基酶和产生突变体的能力将使我们能够直接、明确地确定体内氧化损伤的加工方式。 我们描述了将要实现的三个目标。 1) 使用独特的细胞测定来监测体内病变的修复，我们将鉴定负责基因组整体修复的生物学相关的氧化DNA糖基化酶，并随后使用LC/MS/MS和GC/MS鉴定底物病变。 2）我们将确定一种新型的氧化应激全局传感器短暂关闭复制和转录以响应氧化应激的基因和机制。 3）我们将确定修复和跨损伤合成在处理复制过程中遇到的氧化损伤中的贡献，并识别在存在这些损伤的情况下复制恢复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会导致人类疾病状态，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 损伤在导致人类疾病中的具体作用的理解。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、阿尔茨海默病、肌萎缩侧索硬化症、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正常衰老过程中发挥着重要作用。 确定氧化修复酶的细胞作用以及复制过程中损伤的处理方式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，由于氧化性 DNA 损伤会产生强烈的细胞凋亡信号，因此该研究可能会带来新的化疗模式，包括选择性抑制本研究中确定的修复酶，并结合施用复制抑制剂或抗氧化剂。

项目成果

Completion of DNA replication in Escherichia coli.

在大肠杆菌中完成 DNA 复制。

• DOI: 10.1073/pnas.1415025111
• 发表时间: 2014
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Wendel,BrianM;Courcelle,CharmainT;Courcelle,Justin
• 通讯作者: Courcelle,Justin

UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli.

• DOI: 10.1155/2012/271453
• 发表时间: 2012
• 期刊: Journal of nucleic acids
• 影响因子: 2.3
• 作者: Newton KN;Courcelle CT;Courcelle J
• 通讯作者: Courcelle J