Characterization of the major DNA repair methyltransferase activity in unadapted Escherichia coli and identification of a similar activity in Salmonella typhimurium.
Escherichia coli has two DNA repair methyltransferases (MTases): the 39-kilodalton (kDa) Ada protein, which can undergo proteolysis to an active 19-kDa fragment, and the 19-kDa DNA MTase II. We characterized DNA MTase II in cell extracts of an ada deletion mutant and compared it with the purified 19-kDa Ada fragment. Like Ada, DNA MTase II repaired O6-methylguanine (O6MeG) lesions via transfer of the methyl group from DNA to a cysteine residue in the MTase. Substrate competition experiments indicated that DNA MTase II repaired O4-methylthymine lesions by transfer of the methyl group to the same active site within the DNA MTase II molecule. The repair kinetics of DNA MTase II were similar to those of Ada; both repaired O6MeG in double-stranded DNA much more efficiently than O6MeG in single-stranded DNA. Chronic pretreatment of ada deletion mutants with sublethal (adapting) levels of two alkylating agents resulted in the depletion of DNA MTase II. Thus, unlike Ada, DNA MTase II did not appear to be induced in response to chronic DNA alkylation at least in this ada deletion strain. DNA MTase II was much more heat labile than Ada. Heat lability studies indicated that more than 95% of the MTase in unadapted E. coli was DNA MTase II. We discuss the possible implications of these results for the mechanism of induction of the adaptive response. A similarly active 19-kDa O6MeG-O4-methylthymine DNA MTase was identified in Salmonella typhimurium.
Rebeck
G W
GW
Charles A. Dana Laboratory of Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115.
Smith
C M
CM
Goad
D L
DL
Samson
L
L
D003545:Cysteine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology* / Q000502:physiology; D006147:Guanine / Q000031:analogs & derivatives / Q000032:analysis; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D019853:O(6)-Methylguanine-DNA Methyltransferase; D012486:Salmonella typhimurium / Q000201:enzymology*
D008769:Methylnitronitrosoguanidine; D006147:Guanine; C008449:O-(6)-methylguanine; C008425:S-methylcysteine; D008741:Methyl Methanesulfonate; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003545:Cysteine