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 据报道，一氧化氮（NO）是注射过程中产生的信号分子，据报道诱导 细胞膜的损伤并导致烷基化DNA加合物的积累，例如1，N6-乙烯二烯 （EA），3，N4-乙烯基细胞（EC）。在这个项目中，我们将研究一氧化氮在 炎症和癌症：没有对ALKB家族DNA修复酶的抑制作用。 ALKB蛋白， 已经建立了一组Fe（II）/α-酮戊二酸二加氧酶，以修复DNA烷基病变 直接逆转机制。在真核和原核物种中存在不同的ALKB同源物。九个这样 同源物存在于哺乳动物细胞（ABH1-8和FTO）中。在人类中，ABH2和ABH3被确定为 DNA修复酶，构成针对DNA加合物的最有效的细胞防御。在初步 研究，我们已经表明没有对ALKB，ABH2和ABH3具有强大的抑制作用。电子顺磁性 共振（EPR）光谱研究也表明与ALKB催化中心的Fe（II）离子没有结合， 从而抑制催化O2结合并消除ALKB的修复活性。中心假设 项目是任何不仅诱发烷基DNA损害，而且还会对细胞造成“两倍”损害 抑制ALKB家族DNA修复酶。该项目的重点是研究 DNA修复和在体外和细胞中分子水平的抑制作用。我们将使用三个目标 展示这个目标。在AIM 1中，我们将化学合成含有特定的DNA寡核苷酸 定义位点的烷基化碱，并分离修复酶。然后生化评估修复 体外烷基-DNA病变并确定这些修复和抑制反应的动力学参数。目标 2，我们将测试一氧化氮对大肠杆菌中DNA加合物的复制效率和诱变性的抑制作用 细胞。通过使用放置在确切已知部位的病变，我们计算了绕过病变的细胞动力学 在维修和抑制能力的条件下，聚合酶和病变引起的突变是 系统变化。在AIM 3中，我们将通过EPR光谱进行表征NO-ALKB加合物并准备 小分子模型的复合物用于无反应性研究。从这些实验中获得的知识 将帮助我们了解抑制DNA修复的分子和细胞机制，并提供 开发新策略以预防和克服细胞损伤和肿瘤发生的见解。全面的， 这些研究将表征一氧化氮在炎症和癌症的病理过程中的作用。 再次，DNA损伤是许多疾病的主要发起者，并且拟议研究的完成将 与人类健康有直接相关。