Mhaskar D N, Chang M J, Hart R W, D'Ambrosio S M
Cancer Res. 1981 Jan;41(1):223-9.
This study reports a rapid assay to distinguish depurination from other forms of alkaline-labile lesions induced in DNA by alkylating agents. Covalently closed circular duplex PM2 DNA was treated with various alkylating agents such as N-methyl-N-nitrosourea, dimethyl sulfate, methyl methanesulfonate, N-ethyl-N-nitrosourea, diethyl sulfate, and ethyl methanesulfonate at pH 6.5. Apurinic sites and subsequent strand breaks were introduced by the hydrolysis of the alkylated purines under nondenaturing conditions by heating alkylated DNA at 70 degrees for 1.5 hr with 0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid:KOH (pH 7.4), 0.1 M KCl, 0.01 M MgCl2, 0.0005 M ethylenediaminetetraacetate, 0.05 M glycine, and 0.01 M putrescine. The number of strand breaks produced, representing the alkylated sites at N-3 and N-7 positions of purines, were quantitated by electrophoresis in 1% neutral agarose slab gels. These results were compared with previously reported carcinogenic and mutagenic effects of these compounds, and a correlation between the apurinic sites, the total alkylated sites, and the biological effect of the alkylating agent was determined.
本研究报告了一种快速检测方法，用于区分脱嘌呤作用与烷基化剂诱导DNA产生的其他形式的碱不稳定损伤。将共价闭合环状双链PM2 DNA在pH 6.5条件下用各种烷基化剂处理，如N-甲基-N-亚硝基脲、硫酸二甲酯、甲磺酸甲酯、N-乙基-N-亚硝基脲、硫酸二乙酯和乙磺酸乙酯。在非变性条件下，通过将烷基化DNA在70℃下与0.05 M 4-(2-羟乙基)-1-哌嗪乙磺酸：氢氧化钾（pH 7.4）、0.1 M氯化钾、0.01 M氯化镁、0.0005 M乙二胺四乙酸、0.05 M甘氨酸和0.01 M腐胺一起加热1.5小时，使烷基化嘌呤水解，从而引入脱嘌呤位点和随后的链断裂。通过在1%中性琼脂糖平板凝胶中进行电泳，对代表嘌呤N-3和N-7位烷基化位点产生的链断裂数量进行定量。将这些结果与先前报道的这些化合物的致癌和诱变作用进行比较，并确定脱嘌呤位点、总烷基化位点与烷基化剂生物学效应之间的相关性。
