Partial purification and characterization of a human 3-methyladenine-DNA glycosylase.
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 and ethyl methane-sulfonate as well as methyl methanesulfonate but not in untreated DNA or in DNA alkylated with nitrogen mustard or irradiated with ultraviolet light or X-rays. The glycosylase was free of detectable endonuclease activity in experiments with untreated DNA or DNA exposed to ultraviolet 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 molecular weight of 34,000, is not dependent on divalent metal ions, and shows optimal activity at pH 7.5--8.5.
D000225:Adenine / Q000031:analogs & derivatives; D000478:Alkylation; D002460:Cell Line; D004247:DNA / Q000378:metabolism / Q000528:radiation effects; D045647:DNA Glycosylases; D004279:DNA, Viral; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000201:enzymology; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D013379:Substrate Specificity; D013950:Thymus Gland
D004279:DNA, Viral; D011089:Polydeoxyribonucleotides; D004247:DNA; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D000225:Adenine