isopropyl-thiogalactoside and dimethyl-sulfate

Salmonella typhimurium, compared to Escherichia coli, is deficient in an inducible glycosylase activity harbouring only constitutive glycosylase functions. 3-Methyladenine-DNA glycosylase I encoded by the E. coli tag gene is a constitutively expressed repair enzyme that primarily removes N3-methyladenine but also N3-methylguanine from DNA by glycosylic cleavage in the first step of the base excision repair. In order to investigate in vivo effect of the overexpressed glycosylase I activity on survival capacity and mutation induction in S. typhimurium, and thereby elucidate the significance of both 3-methylpurines in cellular sensitivity to methylating agents (e.g., DMS), we transformed four his- S. typhimurium strains with the plasmid pCY5 carrying the E. coli tag gene under the control of the lac promoter. Although the 3-methyladenine-DNA glycosylase activity in cells carrying pCY5 was only 10-fold higher on exposure to IPTG compared to the TA1535 control strain carrying pUC8, the overexpression of the Tag protein completely suppressed deficiency in an inducible glycosylase activity, rendering cells resistance to toxic effects of DMS. The suppression was not influenced by the nucleotide excision repair pathway since there was no difference in recovered survival among NER-proficient and NER-deficient strains. The yield of mutation induction in the reversion assay was decreased to the level of spontaneous (his-->his+) revertant colonies showing that in the overall population in overexpressed conditions in vivo 3-methyl-guanine, in addition to 3-methyladenine, must have been removed from DNA by the E. coli Tag protein and therefore accounts for the second most important cytotoxic lesion.

Topics: Adenine; DNA Glycosylases; Escherichia coli; Gene Expression Regulation, Bacterial; Genes, Bacterial; Guanine; Isopropyl Thiogalactoside; Mutagens; Mutation; N-Glycosyl Hydrolases; Nitrosourea Compounds; Salmonella typhimurium; SOS Response, Genetics; Sulfuric Acid Esters; Suppression, Genetic; Transformation, Genetic