Two DNA glycosylases in E. coli which release primarily 3-methyladenine

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Abstract
Two enzymes were partially purified from E. coli and designated 3-methyladenine DNA glycosylases I and II. The glycosylase I is that described by Riazuddin and Lindahl [1978]. The apparent MW of glycosylase I is 20,000 and that of II is 27,000. Glycosylase I releases 3-methyladenine (3-MeA) while II releases 3-MeA, 3-methylguanine (3-MeG), 7-methylguanine (7-MeG) and 7-methyladenine (7-MeA). The rate of release of 3-MeA by glycosylase II is 30 times that of 7-MeG. Glycosylase I is missing in mutants tag 1 and tag 2 (Karran, P., et al., 1980). In crude extracts, the 3-MeA activity of II is .apprx. 10% of the total 3-MeA activity. A 50% inactivation at 48.degree. C required 5 min for I and 65 min for II. The apparent Km for 3-MeA residues for glycosylase I was 1.4 .times. 10-8 M. The enzyme was inhibited noncompetitively by 3-MeA with an average apparent Ki of 1.6 mM. The apparent Km for 3-MeA for glycosylase II was 9.2 .times. 10-9 M, and it was not inhibited by 3-MeA. The 3-MeA and 7-MeG activities of the glycosylase II preparation could not be separated by isoelectric focusing, by chromatography on DEAE, Sephadex G-100, phosphocellulose, DNA-cellulose or carboxymethylcellulose or by heating at 50.degree. C. The apparent Km for 7-MeG was 1.1 .times. 10-8 M. Glycosylase II released N1-(carboxyethyl)adenine and N7-(carboxymethyl)guanine from DNA treated with .beta.-[3H]propiolactone but did not release the aflatoxin B-1 adduct at N-7 of guanine.