R15 AREA: Replication in the Presence of Oxidative DNA damage

描述(由申请人提供)： 项目摘要氧化DNA损伤与一系列涉及发育和神经功能进行性丧失的人类疾病状态有关，包括Cockaynes综合征、帕金森综合症和阿尔茨海默病。到目前为止，这是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，最终会损害受损或自然衰老个体的细胞功能或生存能力。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未确定。这部分是因为细胞将一系列酶用于修复和耐受氧化损伤，而这些酶在生化分析中似乎是多余的，部分是因为缺乏细胞分析，这使得在哺乳动物细胞中解决这些问题具有挑战性。这项提议的结果将直接使用大肠杆菌的模式生物来解决这些问题，在大肠杆菌中，复制和氧化DNA修复都高度保守。我们已经在大肠杆菌中建立了独特的细胞分析来监控复制分叉过程，我们快速纯化糖基酶和产生突变的能力将使我们能够直接和最终确定氧化损伤在体内是如何处理的。我们描述了将实现的三个目标。1)使用一种独特的细胞检测方法来监测体内损伤的修复，我们将利用LC/MS/MS和GC/MS鉴定负责基因组整体修复的生物相关的氧化DNA糖基酶，并亚连续地识别底物损伤。2)我们将识别一种新的全球氧化应激传感器瞬时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病变合成在处理复制过程中遇到的氧化损伤方面的贡献，并确定在存在这些损伤的情况下复制恢复过程中出现的中间产物。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加工事件是否会导致人类疾病状态，并可能建议针对这些复制或修复途径的新的治疗方法。 公共卫生相关性： 意义：这个项目的结果将加强我们对氧化DNA损伤在导致人类疾病中所起的具体作用的理解。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、Friedreich共济失调、Fanconi贫血和Cockayne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都发挥着重要作用。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，这项研究可能会导致新的化疗模式，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.