Recombinant Human DNA (Cytosine-5) Methyltransferase

Abstract

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, (CGG⅐CCG) 12 , (m 5 CGG⅐CCG) 12 , and (CGG⅐CCG) 73 but were not linear for (CGG⅐Cm 5 CG) 12 . Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at <20 M 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.The genome of most organisms contains modified nucleotides including N 6 -methyladenine, N 4 -methylcytosine, and C 5 -methylcytosine (m 5 C) 1 (1-3). However, both the biological significance and the types of DNA methylation differ greatly between prokaryotes and eukaryotes. In prokaryotes, most modified bases participate in restriction-modification, a defense mechanism that protects the host from heterologous phage infection (4). In addition, N 6 -methyladenine plays a role in the initiation of DNA replication (5) and in post-replicative methyl-directed mismatch repair (6).In higher eukaryotes, DNA methylation is confined to m 5 C and is implicated in the regulation of development, genomic imprinting (7, 8), X chromosome inactivation, gene expression (9), and retrotransposon inactivation (10 -12). In mammals, the patterns of methylation are inherited from both parental genomes but are erased and reconstructed (de novo methylation) in somatic cells following implantation (13,14). Such patterns are then copied and maintained by hemimethylation of the daughter strands during semiconservative DNA synthesis in S phase (maintenance methylation) (15, 16). However, the mechanisms by which both de novo and maintenance methylation occur remain to be elucidated.Several DNA methyltransferases have been isolated from human and mouse (17-20), but it is not clear whether the de novo and maintenance methylations are carried out by separate proteins in vivo or whether both activities are shared by one or more enzymes (21-26). Nevertheless, a role for in vivo methylation has been established for isoforms of the human DNMT1 gene (27, 28) whose product, DNMT1, me...

Preferential Methylation of Unmethylated DNA by Mammalian de Novo DNA Methyltransferase Dnmt3a

“…Similarly, the stimulatory effect of DNA binding affinity under high concentration of the substrate (40) may account for the slight discrepancy in the estimated substrate preference of Dnmt3a between the ratio under saturating conditions (3.6-fold) and k cat /K m CG under nonsaturating conditions (2.4-fold). Similar to the analyses described in previous reports (14,22,31), secondary plots were generated to analyze the kinetic constants presented in this report. It was confirmed that these values are not significantly different from the results analyzed by the program Sigma Plot/Enzyme Kinetics Module (not shown).…”

“…Mammalian maintenance DNA methyltransferase DNMT1 has been extensively studied biochemically and enzymatically (14,(27)(28)(29)(30)(31). DNMT1 prefers hemimethylated dsDNA and its kinetic constants have been reported (14,28,29).…”

Recombinant Human DNA (Cytosine-5) Methyltransferase

“…⌬501 and DNMT1 with all of the DNA substrates used and the calculation of the steady-state kinetic constants were as described (29). The concentrations of the reactants and those of the products analyzed were always converted to nanomoles or micromoles per liter and therefore are expressed in nM or M, respectively.…”

“…Enzyme preparations were Ͼ95% pure (19). Determination of the Kinetic Constants-We previously developed a velocity equation for DNMT1 (29), based on the King-Altman and Cleland methods (30), that considered the enzyme species bound to the AdoMet and unmethylated DNA substrates but not to the AdoHcy and methylated DNA products (initial forward velocity equation). These conditions are met experimentally when the amount of substrate utilized is negligible compared with its total concentration.…”

“…The synthesis and characterization of these substrates were reported (19,29). Negatively supercoiled pRW3602 (32) was isolated from E. coli strain HB101 and purified by CsCl banding (33).…”