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J Biol Chem, Vol. 274, Issue 46, 33011-33019, November 12, 1999

Recombinant Human DNA (Cytosine-5) Methyltransferase
II. STEADY-STATE KINETICS REVEAL ALLOSTERIC ACTIVATION BY METHYLATED DNA

From the Center for Genome Research, Institute of Biosciences and Technology, Texas A & M University, Texas Medical Center, Houston, Texas 77030-3303 and ^§ New England Biolabs, Beverly, Massachusetts 01915

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 with 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-1 from the imprinted small nuclear ribonucleoprotein-associated polypeptide N, (CGG·CCG)₁₂, (m⁵CGG·CCG)₁₂, and (CGG·CCG)₇₃ but were not linear for (CGG·Cm⁵CG)₁₂. Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at 20 µM AdoMet. Addition of the product (methylated DNA) to unmethylated plasmid DNA increased V_max(app) resulting in mixed stimulation and inhibition. Velocity equations indicated a two-step mechanism as follows: first, activation of DNMT1 by methylated DNA that bound to an allosteric site, and second, the addition of AdoMet 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. We propose that this activation plays a role in vivo in the regulation of maintenance methylation.

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