Mechanism of rat liver DNA methyltransferase interaction with anti-benzo[a]pyrenediol epoxide modified DNA templates.
We investigated the methylation reaction catalyzed by 1500-fold purified rat liver DNA methyltransferase (DMase) on native Micrococcal luteus DNA (ML-DNA) and poly(dC-dG) templates containing covalently bound (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the strongly carcinogenic, principal metabolite of benzo[a]pyrene. Since eukaryotic DNA methyltransferases recognize the dinucleotide 5'd[CG] in DNA as a substrate for methylation, the model polynucleotide poly(dC-dG) was used to study in more detail the mode of interaction and effect on incorporation. With either of these BPDE-modified templates, a progressive inhibition of methylation was correlated with increasing amount of BPDE substitution. The effect of BPDE-dG adducts did not alter the apparent km with respect to the concentration of d[CG] in either unmodified or BPDE-modified poly(dC-dG) (km = 10 microM) but lowered the relative apparent Vmax. In assays in which perturbation by salt of preformed enzyme-DNA complex is measured, no change in the relative stability to either unsubstituted or the carcinogen-modified template was noted, thus, excluding any change in the ionic component of this interaction. However, in competition-type experiments, BPDE-DNA is an inhibitor of the methylation reaction on native DNA. When BPDE-DNA is allowed to interact with the enzyme before the addition of native competitor DNA, the methylation rate is not stimulated, suggesting very tight hydrophobic binding of the enzyme to BPDE-DNA and an inhibition in the dissociation of DMase from the template following a methylation event.(ABSTRACT TRUNCATED AT 250 WORDS)
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D000818:Animals; D001580:Benzopyrenes; D055598:Chemical Phenomena; D002621:Chemistry; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial; D015252:Deoxyribonucleases, Type II Site-Specific; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008837:Micrococcus / Q000032:analysis; D009690:Nucleic Acid Conformation; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D051381:Rats; D013329:Structure-Activity Relationship; D013698:Templates, Genetic
D001580:Benzopyrenes; D004269:DNA, Bacterial; D011089:Polydeoxyribonucleotides; C019530:poly(dC-dG); D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA; D008780:Methyltransferases; D004248:DNA (Cytosine-5-)-Methyltransferases; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases