Transcription-coupled repair of Oxidative DNA damage in vivo

描述（由申请人提供）：阻断转录的DNA损伤可阻止必需基因的表达，导致突变、细胞凋亡或坏死细胞死亡。转录偶联修复是一种细胞过程，通过这种过程，活性基因的转录链比非转录链或整个基因组更快地修复某些形式的DNA损伤。柯凯因综合征患者的特点是发育和神经缺陷，在转录偶联修复过程中存在特异性缺陷。人们普遍推测，氧化dna损伤的转录偶联修复可能是柯凯因综合征潜在发育和神经缺陷的潜在原因，并可能涉及其他涉及神经功能进行性丧失的疾病，如帕金森病和阿尔茨海默病。然而，氧化修复相对于转录的快速动力学，以及活性氧诱导的凋亡级联和停滞的转录机制，使得在哺乳动物细胞中解决这一假设在技术上变得困难，尽管付出了巨大的努力。因此，我们建议在大肠杆菌模型生物中直接验证这一假设，因为大肠杆菌的转录偶联修复和DNA氧化修复过程是高度保守的。我们表明，低复杂性的基因组，充分表征的转录操纵子，以及纯化DNA糖基酶和等基因突变体的使用，使我们能够克服在人类细胞培养中产生的障碍，以检测和明确地回答这个重要问题。我们假设特定的氧化DNA损伤在体内以转录偶联的方式修复。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的影响，而非阻断病变则不会，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定的DNA糖基化酶。为了验证这些假设，我们将1)使用已知底物特异性的纯化DNA糖基酶来测量体内不同氧化DNA损伤的修复动力学；2)检查不同类型的氧化损伤的修复率，8-氧鸟嘌呤，胸腺嘧啶乙二醇等，以确定哪些类型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糖基酶或Mfd的等基因突变体氧化损伤的修复率和RNA合成的恢复。

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.