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.

抽象的 一氧化氮 (NO) 是一种在炎症过程中过度产生的信号分子，据报道可诱导 细胞膜损伤并导致烷基化DNA加合物的积累，例如1,N6-乙烯腺嘌呤 (eA)、3,N4-乙烯胞嘧啶 (eC)。在这个项目中，我们将研究一氧化氮在细胞中的作用的新方面 炎症和癌症：NO 对 AlkB 家族 DNA 修复酶的抑制作用。 AlkB 蛋白， 一组 Fe(II)/α-酮戊二酸依赖性双加氧酶已被建立来修复 DNA 烷基损伤 直接逆转机制。 AlkB 的不同同源物存在于真核和原核物种中；九个这样的 同源物存在于哺乳动物细胞中（ABH1-8 和 FTO）。在人类中，ABH2 和 ABH3 已被确定为 DNA 修复酶并构成针对 DNA 加合物的最有效的细胞防御。在预赛中 研究表明，NO对AlkB、ABH2和ABH3有很强的抑制作用。电子顺磁 共振 (EPR) 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 结合并消除 AlkB 的修复活性。这个假设的中心假设 项目认为，NO 不仅会诱导烷基 DNA 损伤，还会对细胞造成“双重”损伤 抑制 AlkB 家族 DNA 修复酶。该项目的重点是研究之间的关系 DNA 修复和在体外和细胞内分子水平上的 NO 抑制。我们将利用这三个目标 展示这个目标。在目标1中，我们将化学合成含有特定的DNA寡核苷酸 在指定位点烷基化碱基，并分离修复酶。然后通过生化评估修复情况 烷基-DNA 损伤体外并确定这些修复和抑制反应的动力学参数。瞄准 2、测试一氧化氮对大肠杆菌DNA加合物复制效率和致突变性的抑制作用 细胞。通过使用位于确切已知部位的病变，我们通过以下方式计算病变旁路的细胞内动力学： 在修复和抑制能力受到影响的条件下聚合酶和损伤诱导的突变 系统地变化。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复合物。从这些实验中获得的知识 将帮助我们了解 NO 抑制 DNA 修复的分子和细胞机制，并提供 开发新策略来预防和克服细胞损伤和肿瘤发生的见解。全面的， 这些研究将描述一氧化氮在炎症和癌症病理过程中的作用。 DNA 损伤再次成为许多疾病的主要引发因素，完成拟议的研究将 与人体健康有直接关系。