Relative efficiencies of the bacterial, yeast, and human DNA methyltransferases for the repair of O6-methylguanine and O4-methylthymine. Suggestive evidence for O4-methylthymine repair by eukaryotic methyltransferases.
The suicidal inactivation mechanism of DNA repair methyltransferases (MTases) was exploited to measure the relative efficiencies with which the Escherichia coli, human, and Saccharomyces cerevisiae DNA MTases repair O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT), two of the DNA lesions produced by mutagenic and carcinogenic alkylating agents. Using chemically synthesized double-stranded 25-base pair oligodeoxynucleotides containing a single O6MeG or a single O4MeT, the concentration of O6MeG or O4MeT substrate that produced 50% inactivation (IC50) was determined for each of four MTases. The E. coli ogt gene product had a relatively high affinity for the O6MeG substrate (IC50 8.1 nM) but had an even higher affinity for the O4MeT substrate (IC50 3 nM). By contrast, the E. coli Ada MTase displayed a striking preference for O6MeG (IC50 1.25 nM) as compared to O4MeT (IC50 27.5 nM). Both the human and the yeast DNA MTases were efficiently inactivated upon incubation with the O6MeG-containing oligomer (IC50 values of 1.5 and 1.3 nM, respectively). Surprisingly, the human and yeast MTases were also inactivated by the O4MeT-containing oligomer albeit at IC50 values of 29.5 and 44 nM, respectively. This result suggests that O4MeT lesions can be recognized in this substrate by eukaryotic DNA MTases but the exact biochemical mechanism of methyltransferase inactivation remains to be determined.
Sassanfar
M
M
Laboratory of Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115.
Dosanjh
M K
MK
Essigmann
J M
JM
Samson
L
L
D001483:Base Sequence; D002273:Carcinogens; D004249:DNA Damage; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009153:Mutagens; D019853:O(6)-Methylguanine-DNA Methyltransferase; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013941:Thymine / Q000031:analogs & derivatives* / Q000378:metabolism
D002273:Carcinogens; D009153:Mutagens; C021223:O-4-methylthymine; D006147:Guanine; C008449:O-(6)-methylguanine; D008780:Methyltransferases; C040998:O(4)-methylthymine-DNA methyltransferase; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013941:Thymine