Specificity of DNA repair methyltransferases determined by competitive inactivation with oligonucleotide substrates: evidence that Escherichia coli Ada repairs O6-methylguanine and O4-methylthymine with similar efficiency.
DNA repair methyltransferases (MTases) are stoichiometric acceptor molecules that are irreversibly inactivated in the course of removing a methyl group from O6-methylguanine (meG)-DNA or O4-methylthymine (meT)-DNA. A new assay has been developed to determine the relative efficiency of repair of meG and meT. The assay is based on the deprotection of methylated restriction sites in synthetic oligonucleotides and can be used to measure meG repair or meT repair directly. More importantly, relative repair efficiencies can be measured in competition experiments, using each of the methylated oligomers in turn as an inhibitor of repair for the other. Relative repair rates are determined by numerical solution of the coupled rate equations that describe this competition to the experimental data. We find that the human MTase repairs meT about 35-fold less well than meG, qualitatively similar to earlier studies. Contrary to previous reports, however, we find that Escherichia coli Ada repairs meG and meT with nearly equal efficiency. This finding, in conjunction with other recent reports, may indicate that low meT repair is a relatively unusual characteristic of the human homolog.
Paalman
S R
SR
Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Sung
C
C
Clarke
N D
ND
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D008780:Methyltransferases / Q000378:metabolism*; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010446:Peptide Fragments / Q000378:metabolism; D013941:Thymine / Q000031:analogs & derivatives / Q000378:metabolism; D014157:Transcription Factors
C086794:Ada-C protein, E coli; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D014157:Transcription Factors; C021223:O-4-methylthymine; D006147:Guanine; C008449:O-(6)-methylguanine; D008780:Methyltransferases; C040998:O(4)-methylthymine-DNA methyltransferase; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013941:Thymine