Tudek B, Graziewicz M, Kazanova O, Zastawny T H, Obtułowicz T, Laval J
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa.
Acta Biochim Pol. 1999;46(3):785-99.
The most abundant lesion formed in DNA upon modification with methylating agents 7-methylguanine, under alkaline conditions is converted into 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeGua). We have previously shown that treatment of dimethylsulfate methylated DNA with NaOH creates mutagenic base derivatives leading to a 60-fold increase in the frequency of A-->G transitions and a 2-3-fold increase of G-->T and G-->C transversions. We have analyzed which lesions lead to these mutations. We compared mutagenic spectra in the lacZ gene of M13mp18 phage DNA modified with dimethylsulfate and NaOH after selective elimination of damaged bases from molecules used for transfection into SOS-induced E. coli. Partial elimination of Fapy-7MeGua from phage DNA performed by its digestion with formamidopyrimidine-DNA glycosylase resulted in a 2-3-fold decrease of G-->T and G-->C transversions. Selective depurination of methylated bases (9 h, 37 degrees C, pH 7.0) resulting in almost complete loss of 7MeAde as demonstrated by HPLC analysis of [3H]MNU alkylated phage DNA used as a probe, caused a dramatic, 9-fold decrease of A-->G transitions. Alkali-catalysed rearrangement of 7MeAde was followed by HPLC analysis of [3H]MNU alkylated poly(A) and poly(dA). After incubation of these oligonucleotides in NaOH, 7MeAde disappeared from both chromatograms, but only in polyA, 2 new peaks migrating with retention time different from that of 1MeAde, 3MeAde or 7MeAde were detected, suggesting formation of two rotameric forms of Fapy-7MeAde as observed for Fapy-7MeGua. Thus the miscoding lesion, giving rise to A-->G transitions derived from 7MeAde was Fapy-7MeAde. Fapy-7MeGua was at least an order of magnitude less mutagenic, but in SOS-induced cells it gave rise to G-->T and G-->C transversions.
在用甲基化试剂修饰DNA时形成的最丰富的损伤产物7 - 甲基鸟嘌呤，在碱性条件下会转化为2,6 - 二氨基 - 4 - 羟基 - 5N - 甲基 - 甲酰胺基嘧啶（Fapy - 7MeGua）。我们之前已经表明，用氢氧化钠处理硫酸二甲酯甲基化的DNA会产生诱变碱基衍生物，导致A→G转换频率增加60倍，G→T和G→C颠换频率增加2 - 3倍。我们分析了哪些损伤会导致这些突变。我们比较了在用硫酸二甲酯和氢氧化钠修饰的M13mp18噬菌体DNA的lacZ基因中的诱变谱，这些DNA在用于转染到SOS诱导的大肠杆菌之前，先对损伤碱基进行了选择性去除。通过用甲酰胺基嘧啶 - DNA糖基化酶消化噬菌体DNA来部分去除Fapy - 7MeGua，导致G→T和G→C颠换减少2 - 3倍。甲基化碱基的选择性脱嘌呤（9小时，37℃，pH 7.0）导致7MeAde几乎完全丢失，这通过对用作探针的[³H]MNU烷基化噬菌体DNA进行HPLC分析得以证明，这导致A→G转换急剧下降9倍。对[³H]MNU烷基化的聚（A）和聚（dA）进行HPLC分析，跟踪7MeAde的碱催化重排。在这些寡核苷酸在氢氧化钠中孵育后，7MeAde从两个色谱图中消失，但仅在聚A中检测到两个新峰，其保留时间与1MeAde、3MeAde或7MeAde不同，这表明形成了两种Fapy - 7MeAde的旋转异构体形式，就像在Fapy - 7MeGua中观察到的那样。因此，导致源自7MeAde的A→G转换的错配损伤是Fapy - 7MeAde。Fapy - 7MeGua的诱变能力至少低一个数量级，但在SOS诱导的细胞中它会导致G→T和G→C颠换。
