Detection of DNA sites damaged by 1-(4-amino-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) using a DNA sequencing procedure

Abstract

A DNA sequencing technique was applied to the highly reiterated DNA from HeLa S 3 cells in order to detect DNA damage induced by the antitumor drug, 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). A DNA reiterated fragment of 92 base pairs (bp) was isolated by gel electrophoresis after Eco RI and Eco RI ∗ restriction endonuclease digestion. In the defined sequence of the 92 bp fragment, ACNU caused damage and modifications primarily at guanine moieties, leading to alkali-labile sites as determined by subsequent piperidine reaction on an extended Maxam-Gilbert sequencing gel. These results indicate that guanine moieties in double-stranded DNA are preferentially vulnerable to ACNU over other base moieties.