Inhibitory Effect of Nitric Oxide on DNA Repair Enzymes

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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.