Partial purification and characterization of a human 3-methyladenine-DNA glycosylase

Abstract

A DNA glycosylase was purified about 30-fold from cultured human lymphoblasts (CCRF-CEM line) and was found to cleave 3-methyladenine from DNA alkylated with methyl methanesulfonate. The enzyme did not promote the release of 1-methyladenine, 7-methyladenine or 7-methylguanine from DNA nor did it act on denatured methylated DNA. It produced apurinic sites in DNA alkylated with N-methyl-N-nitrosourea, ethyl methanesulfonate and methyl methanesulfonate but not in untreated DNA or in DNA alkylated with nitrogen mustard or irradiated with UV light or X-rays. The glycosylase was free of detectable endonuclease activity in experiments with untreated DNA or DNA exposed to UV light; low levels of endonuclease activity, obtained when X-irradiated, alkylated or depurinated DNA was the substrate, were attributed to contaminant apurinic endonuclease activity. This 3-methyladenine-DNA glycosylase has an estimated MW of 34,000, is not dependent on divalent metal ions and shows optimal activity at pH 7.5-8.5.