DNA cytosine C5 methyltransferase Dnmt1: catalysis-dependent release of allosteric inhibition.
We followed the cytosine C(5) exchange reaction with Dnmt1 to characterize its preference for different DNA substrates, its allosteric regulation, and to provide a basis for comparison with the bacterial enzymes. We determined that the methyl transfer is rate-limiting, and steps up to and including the cysteine-cytosine covalent intermediate are in rapid equilibrium. Changes in these rapid equilibrium steps account for many of the previously described features of Dnmt1 catalysis and specificity including faster reactions with premethylated DNA versus unmethylated DNA, faster reactions with DNA in which guanine is replaced with inosine [poly(dC-dG) vs poly(dI-dC)], and 10-100-fold slower catalytic rates with Dnmt1 relative to the bacterial enzyme M.HhaI. Dnmt1 interactions with the guanine within the CpG recognition site can prevent the premature release of the target base and solvent access to the active site that could lead to mutagenic deamination. Our results suggest that the beta-elimination step following methyl transfer is not mediated by free solvent. Dnmt1 shows a kinetic lag in product formation and allosteric inhibition with unmethylated DNA that is not observed with premethylated DNA. Thus, we suggest the enzyme undergoes a slow relief from allosteric inhibition upon initiation of catalysis on unmethylated DNA. Notably, this relief from allosteric inhibition is not caused by self-activation through the initial methylation reaction, as the same effect is observed during the cytosine C(5) exchange reaction in the absence of AdoMet. We describe limitations in the Michaelis-Menten kinetic analysis of Dnmt1 and suggest alternative approaches.
Svedruzić
Zeljko M
ZM
Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, USA.
Reich
Norbert O
NO
D000494:Allosteric Regulation; D000818:Animals; D002384:Catalysis; D045744:Cell Line, Tumor; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000737:chemistry; D015257:DNA-Cytosine Methylases / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology; D051379:Mice; D011089:Polydeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014316:Tritium / Q000737:chemistry / Q000378:metabolism
D004269:DNA, Bacterial; D011089:Polydeoxyribonucleotides; D014316:Tritium; C031156:poly d(I-C); C019530:poly(dC-dG); D012436:S-Adenosylmethionine; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619381:Dnmt1 protein, mouse