Cloning of Escherichia coli genes encoding 3-methyladenine DNA glycosylases I and II.
We have constructed two recombinant plasmids which harbour functions involved in DNA repair of alkylation damage in Escherichia coli. One plasmid carries the tag+ gene encoding 3-methyladenine DNA glycosylase I while the other carries alkA+ encoding 3-methyladenine DNA glycosylase II. The plasmids were isolated from plasmid stocks carrying total cellular DNA by selection for their ability to complement the methylmethanesulphonate(MMS)-sensitive phenotype of an E. coli mutant (tag ada) deficient in both 3-methyladenine DNA glycosylases I and II. Both plasmids increase the plating efficiency of such a mutant on methylmethanesulphonate plates by a factor of more than 10(5). The tag gene is located on a 6 (kbp) HindIII fragment, and the presence of the tag plasmid in the cells results in 15-fold overproduction of 3-methyladenine DNA glycosylase I. The other plasmid restores 3-methyladenine DNA glycosylase II deficiency in alkA mutant cells, and results in 3-fold overproduction of this enzyme after alkylation induction. The induction is ada+-dependent and we conclude that this plasmid contains the structural gene for 3-methyladenine DNA glycosylase II, including its control region responding to alkylation induction. However, the plasmid does not complement fully the MMS-sensitive phenotype of alkA mutants which suggests that the plasmid may not include the entire alkA operon.
D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004260:DNA Repair; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D008745:Methylation; D009699:N-Glycosyl Hydrolases / Q000235:genetics*; D010957:Plasmids
D004262:DNA Restriction Enzymes; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases