Purification and characterization of Escherichia coli formamidopyrimidine-DNA glycosylase that excises damaged 7-methylguanine from deoxyribonucleic acid

Abstract

A DNA glycosylase excises 7-methylguanines with alkali-opened imidazole rings (formamidopyrimidines) from DNA was purified more than 8000-fold from E. coli cell extracts. The enzyme does not cleave 3-methyladenine, uracil and intact 7-methylguanine from DNA. In assays containing pyrimidine analogs like oxauracil, 2,4,6-triaminopyrimidine, 2,5,6-triamino-2-hydroxypyrimidine sulfate, formamidopyrimidine and 5-nitroso-2,4,6-triaminopyrimidine, only the last 2 compounds showed end product inhibition of the enzyme. The enzyme was named formamidopyrimidine-DNA glycosylase. It has a MW of 30,000 and a Stokes radius of 26.4 .ANG.. The enzyme prefers double-stranded to single-stranded DNA and is stimulated by the presence of 0.1 M KCl in the reaction mixture.