Inhibitory Effect of Nitric Oxide on DNA Repair Enzymes

一氧化氮对DNA修复酶的抑制作用

• 批准号: 9232253
• 负责人: Deyu Li
• 金额: $ 41.1万
• 依托单位: UNIVERSITY OF RHODE ISLAND
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9232253
• 关键词: Area; Basic Science; Binding; Biochemical; Biochemical Genetics; Biological; Biomimetics; Biophysics; Bypass; Cell Cycle Kinetics; Cell membrane; Cells; Chemicals; Collaborations; Complex; Core Facility; DNA; DNA Adducts; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA Repair Inhibition; DNA lesion; Dioxygenases; Disease; Education; Electron Spin Resonance Spectroscopy; Environment; Enzymes; Escherichia coli; Family; Genome; Goals; Health; Human; Immune response; Impairment; In Vitro; Induced Mutation; Inflammation; Inflammation Process; Institutes; Ions; Kinetics; Knowledge; Laboratories; Lead; Lesion; Malignant Neoplasms; Mammalian Cell; Massachusetts; Measures; Metalloproteins; Modeling; Molecular; Molecular Models; Nitric Oxide; Nucleic Acids; Oligonucleotides; Pathologic Processes; Pharmacologic Substance; Pharmacy facility; Polymerase; Procollagen-Proline Dioxygenase; Property; Protein Family; Proteins; Reaction; Reporting; Research; Research Infrastructure; Role; Science; Signaling Molecule; Site; Students; Synthesis Chemistry; Technology; Testing; Therapeutic; Training; Universities; Work; adduct; alpha ketoglutarate; authority; base; biological systems; carcinogenesis; cell injury; college; cytotoxic; design; enzyme activity; experimental study; genome integrity; histone demethylase; improved; inhibitor/antagonist; insight; instrumentation; metal complex; molecular modeling; prevent; programs; repair enzyme; repaired; small molecule; spectroscopic survey; student mentoring; tool; tumorigenesis; weapons; working group

Abstract Nitric oxide (NO), a signaling molecule over produced in the inflammation process, has been reported to induce damage of cell membrane and lead to the accumulation of alkylated DNA adducts, such as 1,N6-ethenoadenine (eA), 3,N4-ethenocytosine (eC). In this project, we will study a new aspect of nitric oxide’s cellular role in inflammation and cancer: the inhibitory effect of NO on the AlkB family DNA repair enzymes. The AlkB proteins, a group of Fe(II)/α-ketoglutarate-dependent dioxygenases, have been established to repair DNA alkyl lesions by a direct reversal mechanism. Different homologs of AlkB exist in eukaryotic and prokaryotic species; nine such homologs exist in mammalian cells (ABH1-8 and FTO). In humans, ABH2 and ABH3 have been identified as DNA repair enzyme and constitute the most effective cellular defense against DNA adducts. In the preliminary study, we have shown that NO has strong inhibitory effect on AlkB, ABH2 and ABH3. Electron paramagnetic resonance (EPR) spectroscopic studies also showed NO binds to the Fe(II) ion in the catalytic center of AlkB, thus inhibiting the catalytic O2 binding and abolishing the repair activity of AlkB. The central hypothesis of this project is that NO delivers a “two-fold” damage to the cell by not only inducing alkyl DNA damages but also suppressing the AlkB family DNA repair enzymes. The focus of this project is to study the relationship between DNA repair and NO inhibition at molecular level both in vitro and in cell. We will use the three aims to demonstrate this goal. In Aim 1, we will chemically synthesize DNA oligonucleotides containing specific alkylated bases at defined sites, and isolate the repair enzymes. And then biochemically evaluate the repair of alkyl-DNA lesions in vitro and determine the kinetic parameters of those repair and inhibitory reactions. In Aim 2, we will test nitric oxide’s inhibitory effect on replication efficiency and mutagenicity of DNA adducts in E. coli cells. By using lesions placed at the exact known sites, we calculate the in cell kinetics of lesion bypass by polymerases and lesion-induced mutation under conditions whereby the repair and inhibition capacity are systematically varied. In Aim 3, we will characterize the NO-AlkB adduct by EPR spectroscopy and prepare small molecule model complexes for NO-AlkB reactivity studies. The knowledge gained from those experiments will help us understand the molecular and cellular mechanisms of NO inhibition on DNA repair and provide insights for developing new strategy to prevent and overcome the cellular damage and tumorigenesis. Overall, these studies will characterize a role of nitric oxide in the pathological processes of inflammation and cancer. Once again, DNA damage is a primary initiator of many diseases and completion of the proposed studies will have direct relevance to human health.

摘要 一氧化氮(-NO)是炎症过程中过量产生的一种信号分子，据报道可诱导 破坏细胞膜并导致烷化DNA加合物的积累，如1，N6-乙烯腺嘌呤 (EA)，3，N4-乙胞嘧啶(EC)。在这个项目中，我们将研究一氧化氮在细胞中作用的一个新方面 炎症与癌症：-NO对AlkB家族dna修复酶的抑制作用。AlkB蛋白， 已经建立了一组依赖Fe(II)/α-酮戊二酸的双加氧酶，用于修复DNA烷基损伤 一种直接反转机制。AlkB的不同同源物存在于真核和原核物种中； 哺乳动物细胞(ABH1-8和FTO)中存在同源基因。在人类中，ABH2和ABH3已被鉴定为 DNA修复酶，并构成对DNA加合物最有效的细胞防御。在预赛中 研究表明，-NO对碱性磷酸酶B、ABH2和ABH3均有较强的抑制作用。电子顺磁 共振光谱研究还表明，NO与AlkB催化中心的Fe(II)离子结合， 从而抑制催化O2结合，取消AlkB的修复活性。这一点的中心假设是 项目是，-NO不仅通过诱导烷基dna损伤，而且还通过诱导 抑制AlkB家族DNA修复酶。本课题的研究重点是研究二者之间的关系。 DNA修复和在体外和细胞内均不受分子水平的抑制。我们将利用这三个目标来 演示这一目标。在目标1中，我们将化学合成含有特定基因的DNA寡核苷酸。 在指定的位点上烷化碱基，并分离修复酶。然后用生化方法评估修复情况 并测定这些修复和抑制反应的动力学参数。在AIM 2、检测一氧化氮对DNA加合物在大肠杆菌中复制效率和致突变性的抑制作用 细胞。通过使用放置在确切已知位置的病变，我们计算了病变旁路的细胞内动力学。 聚合酶和损伤诱导的突变，其中修复和抑制能力是 系统地变化的。在目标3中，我们将通过EPR光谱表征NO-AlkB加合物，并制备 用于NO-AlkB反应性研究的小分子模型络合物。从这些实验中获得的知识 将有助于我们了解NO抑制DNA修复的分子和细胞机制，并提供 对开发预防和克服细胞损伤和肿瘤发生的新策略的见解。总的来说， 这些研究将确定一氧化氮在炎症和癌症病理过程中的作用。 再一次，DNA损伤是许多疾病的主要诱因，完成拟议的研究将 与人类健康有直接关系。