Borys E, Kuśmierek J T
Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw.
Acta Biochim Pol. 1998;45(2):579-86.
The combined action of glycosylases and abasic site-specific endonucleases on damaged bases in DNA results in single strand breaks. In plasmid DNA, as a consequence, the covalently closed circular (ccc) form is converted to the open circular (oc) form, and this can be quantitated by agarose gel electrophoresis. We studied DNA lesions sensitive to E. coli 3-methyladenine-DNA glycosylase II (AlkA) and cloned human N-alkylpurine-DNA glycosylase (ANPG-40) which are known to excise alkylated bases and etheno adducts. pBR322 and pAlk10 plasmids not pretreated with mutagens were cleaved by both glycosylases in the presence of enzymes possessing endonucleolytic activity, which indicates that plasmids contain unknown, endogenously formed adducts. Plasmids pretreated with chloroacetaldehyde, a mutagen forming etheno adducts, exhibited enhanced sensitivity to both glycosylases. Adducts formed by acrolein and croton aldehyde were excised by AlkA, but not by ANPG-40, whereas malondialdehyde adducts were not excised by either glycosylase. Bulky p-benzochinone adducts were not excised by AlkA, however, the plasmid pretreated with this mutagen was incised by endonucleases, possibly without prior generation of an abasic site. These examples show that examination of conformational changes of plasmid DNA can be taken advantage of to study the specificity of N-alkylpurine-DNA-glycosylases.
糖基化酶和无碱基位点特异性核酸内切酶对DNA中受损碱基的联合作用会导致单链断裂。因此，在质粒DNA中，共价闭合环状（ccc）形式会转变为开环（oc）形式，这可以通过琼脂糖凝胶电泳进行定量分析。我们研究了对大肠杆菌3-甲基腺嘌呤-DNA糖基化酶II（AlkA）和克隆的人N-烷基嘌呤-DNA糖基化酶（ANPG-40）敏感的DNA损伤，已知这两种酶可切除烷基化碱基和乙烯基加合物。未用诱变剂预处理的pBR322和pAlk10质粒在具有核酸内切酶活性的酶存在下会被这两种糖基化酶切割，这表明质粒中含有未知的内源性形成的加合物。用形成乙烯基加合物的诱变剂氯乙醛预处理的质粒对这两种糖基化酶的敏感性增强。丙烯醛和巴豆醛形成的加合物可被AlkA切除，但不能被ANPG-40切除，而丙二醛加合物则不能被这两种糖基化酶切除。大体积的对苯醌加合物不能被AlkA切除，然而，用这种诱变剂预处理的质粒会被核酸内切酶切割，可能无需事先产生无碱基位点。这些例子表明，可以利用质粒DNA构象变化的检测来研究N-烷基嘌呤-DNA-糖基化酶的特异性。
