Cupric ion ascorbate hydrogen peroxide-induced DNA damage: DNA-bound copper ion primarily induces base modifications
Cupric ion ascorbate hydrogen peroxide-induced DNA damage: DNA-bound copper ion primarily induces base modifications
复制标题
DOI：
10.1016/0891-5849(96)00037-8
复制
发表时间：
1996-01-01
影响因子：
7.4
通讯作者：
Akman, SA
中科院分区：
文献类型：
--
作者：
Drouin, R;Rodriguez, H;Akman, SA
同时测定了Cu（II）/抗坏血酸/H2 O2在体外纯化的人基因组DNA中产生的DNA链断裂和DNA碱基修饰的动力学。通过用大肠杆菌酶Nth蛋白（修饰的嘧啶）和Fpg蛋白（修饰的嘌呤）切割来确定修饰的碱基。通过中性乙二醛凝胶电泳定量Nth或Fpg蛋白消化前（坦率的链断裂）和后（修饰的碱基）的单链损伤频率。需要对通过标准蛋白酶K消化/苯酚提取纯化的EDTA处理的基因组DNA进行透析，以去除低分子量物质，可能是过渡金属离子和金属离子螯合剂，这些物质支持在抗坏血酸盐+H2 O2存在且不补充铜离子的情况下发生明显的链断裂。在此基础上，我们建立了Cu（Ⅱ）+抗坏血酸+H_2O_2引起的DNA损伤反应的动力学模型。模型中的主要新假设是DNA碱基修饰完全由DNA结合的Cu（Ⅰ）引起，而非DNA结合的Cu（Ⅰ）引起DNA链断裂。用已发表的速率常数对模型进行了计算机模拟。计算机模拟定量预测：（1）H2 O2降解速率，这是使用H2 O2敏感电极测量的，（2）在反应期间DNA链断裂和修饰碱基积累的线性，（3）修饰碱基积累速率，和（4）修饰碱基和frank链断裂产生对初始Cu（II）浓度的依赖性。模拟显着高估了坦率的链断裂积累的速度，这表明，无论是最终的氧化物质，攻击糖-磷酸骨架是一个反应性较低的物种比模型中使用的羟基自由基和/或一个身份不明的羟基自由基清除物种是存在于反应中。我们的实验数据与铜离子-DNA相互作用的模型一致，其中DNA结合的Cu（I）主要介导DNA碱基修饰，而非结合的Cu（I）主要介导坦率的链断裂产生。
The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which supported frank strand breaks in the presence of ascorbate + H2O2 without supplemental copper ions. We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate + H2O2 The principal new assumption in our model was that DNA base modifications were caused exclusively by DNA-bound Cu(I) and frank strand breaks by non-DNA-bound Cu(I). The model was simulated by computer using published rate constants. The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand break production on initial Cu(II) concentration. The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavening species was present in the reactions. Our experimental data are consistent with a model of copper ion-DNA interaction in which DNA-bound Cu(I) primarily mediates DNA base modifications and nonbound Cu(I) primarily mediates frank strand break production.