DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants
DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants
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DOI：
10.1074/jbc.m806809200
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发表时间：
2008-12-19
影响因子：
4.8
通讯作者：
Dedon, Peter C.
中科院分区：
文献类型：
--
作者：
Margolin, Yelena;Shafirovich, Vladimir;Dedon, Peter C.
DNA序列背景已经成为许多氧化剂造成的核苷酸碱基损伤位置和数量的关键决定因素。然而，强氧化剂如电离辐射和Fe2+-EDTA/H_2O_2的Fenton化学引起的碱基和2-脱氧核糖损伤的复杂性给确定DNA碱基损伤的位置和序列背景对损伤化学的影响提出了挑战。为了解决这个问题，我们开发了一种基于凝胶的方法，通过利用大肠杆菌外切酶III来去除含有直接链断裂和碱性位点的片段，从而定量测定氧化DNA中的核苷酸碱基损伤。该方法的严密性在光氧化核黄素和亚硝基过氧碳酸酯氧化鸟嘌呤的研究中得到了验证，对于这些研究，序列背景的不同影响已经通过其他方法(Margolin，Y.，Cloutier，J.F.，Shafirovich，V.，Geacintov，N.E.和Dedon，P.C.(2006)NAT)得到了验证。化学。比奥尔。2,365-366)。使用包含鸟嘌呤所有可能的三核苷酸序列上下文的双链寡核苷酸，该方法被用来评估DNA序列上下文在与伽马辐射和Fe2+-EDTA/H_2O_2相关的羟基自由基诱导的鸟嘌呤氧化中的作用。结果表明，羟基自由基氧化G与核黄素介导的光催化单电子氧化G既有异同，也有相似之处，这与羟基自由基氧化生成鸟嘌呤自由基阳离子的优势相一致。尽管羟基自由基产生的G氧化相对量与序列特异性电离能的相关性比核黄素产生的G氧化更弱，但在G的两个和三个碱基范围内，羟基自由基发生器和核黄素产生的损伤在位置上表现出偏向，这与电子转移在确定损伤产物位置中的作用是一致的。此外，伽马辐射和Fe2+-EDTA/H_2O_2对不同损伤产物的比例的影响相对较小，尽管含有GT的序列背景在损伤化学(甲酰胺并嘧啶DNA糖基酶/哌啶比率)上表现出微妙的偏差。总体而言，这些结果与已知的羟基自由基氧化鸟嘌呤的化学结果一致，表明电荷迁移在决定DNA中羟基自由基介导的鸟嘌呤氧化损伤的位置和化学方面起到的作用相对较小。
DNA sequence context has emerged as a critical determinant of the location and quantity of nucleobase damage caused by many oxidizing agents. However, the complexity of nucleobase and 2-deoxyribose damage caused by strong oxidants such as ionizing radiation and the Fenton chemistry of Fe2+-EDTA/H2O2 poses a challenge to defining the location of nucleobase damage and the effects of sequence context on damage chemistry in DNA. To address this problem, we developed a gel-based method that allows quantification of nucleobase damage in oxidized DNA by exploiting Escherichia coli exonuclease III to remove fragments containing direct strand breaks and abasic sites. The rigor of the method was verified in studies of guanine oxidation by photooxidized riboflavin and nitrosoperoxycarbonate, for which different effects of sequence context have been demonstrated by other approaches (Margolin, Y., Cloutier, J. F., Shafirovich, V., Geacintov, N. E., and Dedon, P. C. (2006) Nat. Chem. Biol. 2, 365-366). Using duplex oligodeoxynucleotides containing all possible three-nucleotide sequence contexts for guanine, the method was used to assess the role of DNA sequence context in hydroxyl radical-induced guanine oxidation associated with gamma-radiation and Fe2+-EDTA/H2O2. The results revealed both differences and similarities for G oxidation by hydroxyl radicals and by one-electron oxidation by riboflavin-mediated photooxidation, which is consistent with the predominance of oxidation pathways for hydroxyl radicals other than one-electron oxidation to form guanine radical cations. Although the relative quantities of G oxidation produced by hydroxyl radicals were more weakly correlated with sequence-specific ionization potential than G oxidation produced by riboflavin, damage produced by both hydroxyl radical generators and riboflavin within two- and three-base runs of G showed biases in location that are consistent with a role for electron transfer in defining the location of the damage products. Furthermore, both gamma-radiation and Fe2+-EDTA/H2O2 showed relatively modest effects of sequence context on the proportions of different damage products sensitive to E. coli formamidopyrimidine DNA glycosylase and hot piperidine, although GT-containing sequence contexts displayed subtle biases in damage chemistry (formamidopyrimidine DNA glycosylase/piperidine ratio). Overall, the results are consistent with the known chemistry of guanine oxidation by hydroxyl radical and demonstrate that charge migration plays a relatively minor role in determining the location and chemistry of hydroxyl radical-mediated oxidative damage to guanine in DNA.