R15 AREA: Replication in the Presence of Oxidative DNA damage

描述（由申请人提供）： 氧化性DNA损伤与一系列人类疾病状态相关，包括发育和神经功能的进行性丧失，包括Cockaynes综合征，帕金森病和阿尔茨海默病。它是迄今为止细胞遇到的最常见的损伤形式，并且人们广泛推测，疾病是损伤和突变逐渐积累的结果，这些损伤和突变最终损害细胞功能或修复受损或自然老化个体的生存能力。然而，尽管有这些关联，氧化损伤在体内复制过程中处理的细胞机制在很大程度上仍然没有得到表征。在某种程度上，这是因为细胞将一系列酶用于修复和耐受氧化损伤，这些酶在生化测定中显得多余，部分原因是缺乏细胞测定，这使得在哺乳动物细胞中解决这些问题具有挑战性。该提案的结果将直接使用大肠杆菌的模式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。我们已经建立了独特的细胞分析在E。大肠杆菌监测复制叉加工，我们的能力，快速纯化糖基化酶和产生突变体将使我们能够直接和明确地确定如何氧化损伤在体内处理。 我们描述了三个目标，将实现。1)利用独特的细胞分析方法监测体内损伤修复，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，并使用LC/MS/MS和GC/MS间接鉴定底物损伤。2）我们将确定一种新的氧化应激全球传感器的基因和机制，通过这种基因和机制短暂唐斯复制和转录，以响应氧化应激。应力3)我们将确定的贡献，修复和translesion合成在处理氧化损伤复制过程中遇到的，并确定在这些病变的存在下，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受损的处理事件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： 重要性：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特定作用的理解。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损伤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损伤产生强烈的细胞凋亡信号，该研究可能会导致新的化疗模式，包括选择性抑制本研究中鉴定的修复酶，并联合使用复制抑制剂或抗氧化剂。

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.