Recombinant human DNA (cytosine-5) methyltransferase II. Steady-state kinetics reveal allosteric activation by methylated DNA

Initial velocity determinations were conducted with human DNA (cytosine-5) methyltransferase (DNMT1) on unmethylated and hemimethylated DNA templates in order to assess the mechanism of the reaction. Initial velocity data wit h DNA and S-adenosylmethionine (AdoMet) as variable substrates and product inhibition studies with methylated DNA and S-adenosylhomocysteine (AdoHcy) were obtained and evaluated as double-reciprocal plots. These relationships were linear for plasmid DNA, exon-l from the imprinted small nuclear ribon ucleoprotein-associated polypeptide N, (CGG . CCG)(12), (m(5)CGG . CCG)(12) , and (CGG . CCG)(73) but were not linear for (CGG . Cm(5)CG)(12). Inhibiti on by AdoHcy was apparently competitive versus AdoMet and uncompetitive/non competitive versus DNA at less than or equal to 20 mu M AdoMet. Addition of the product (methylated DNA) to unmethylated plasmid DNA increased V-max(a pp) resulting in mixed stimulation and inhibition. Velocity equations indic ated a two-step mechanism as follows: first, activation of DNMT1 by methyla ted DNA that bound to an allosteric site, and second, the addition of AdoMe t and DNA to the catalytic site. The preference of DNMT1 for hemimethylated DNA may be the result of positive cooperativity of AdoMet binding mediated by allosteric activation by the methylated CG steps. Fire propose that thi s activation plays a role in vivo in the regulation of maintenance methylat ion.