Purification and properties of the alkylation repair DNA glycosylase encoded the MAG gene from Saccharomyces cerevisiae.
The MAG gene of Saccharomyces cerevisiae encodes an alkylation repair DNA glycosylase whose sequence is homologous to the AlkA DNA glycosylase from Escherichia coli. To investigate the biochemical properties of MAG in comparison to AlkA, MAG was expressed in E. coli and purified to electrophoretic homogeneity. N-Terminal sequencing of the purified protein identified the translational start site which corresponded to that predicted previously from the nucleotide sequence. Polyclonal antibodies raised against MAG inhibited the enzymatic activity of MAG, but not that of AlkA, and vice versa, implying that the structures of the active site regions of these enzymes are antigenic, but sufficiently different to have different epitopes. Kinetic analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea and [3H]dimethyl sulfate showed that MAG was as effective as AlkA in removing 3-methyladenine, 7-methylguanine, and 7-methyladenine. However, the purified MAG enzyme did not catalyze the excision of O2-methylthymine, which is a major substrate for AlkA. Furthermore, 3-methylguanine was excised 20-40 times more slowly by MAG than by AlkA. The kinetics of 3-methylguanine excision by MAG were found to be similar to the low rate of 3-methylguanine excision catalyzed by 3-methyladenine DNA glycosylase I (Tag) of E. coli. Expression of MAG in alkA mutant cells did not effectively restore alkylation resistance of the mutant as did AlkA itself. It thus appears that MAG is a less versatile enzyme than AlkA in spite of the sequence relationship and may have a similar function in yeast as the nonhomologous Tag enzyme in E. coli.
Bjørås
M
M
Biotechnology Centre, University of Oslo, Blindern, Norway.
Klungland
A
A
Johansen
R F
RF
Seeberg
E
E
D000478:Alkylation; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D014170:Transformation, Genetic
D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases