Recombinant human DNA (cytosine-5) methyltransferase - III. Allosteric control, reaction order, and influence of plasmid topology and triplet repeat length on methylation of the fragile X CGG center dot CCG sequence

Steady-state kinetic analyses revealed that the methylation reaction of the human DNA (cytosine-5) methyltransferase 1 (DNMT1) is repressed by the N-t erminal domain comprising the first 501 amino acids, and that repression is relieved when methylated DNA binds to this region. DNMT1 lacking the first 501 amino acids retains its preference for hemimethylated DNA, The methyla tion reaction proceeds by a sequential mechanism, and either substrate (S-a denosyl-L-methionine and unmethylated DNA) may be the first to bind to the active site. However, initial binding of S-adenosyl-L-methionine is preferr ed. The binding affinities of DNA for both the regulatory and the catalytic sites increase in the presence of methylated CpG; dinucleotides and vary c onsiderably (more than one hundred times) according to DNA sequence. DNA to pology strongly influences the reaction rates, which increased with increas ing negative superhelical tension. These kinetic data are consistent with t he role of DNMT1 in maintaining the methylation patterns throughout develop ment and suggest that the enzyme may be involved in the etiology of fragile X, a syndrome characterized by de novo methylation of a greatly expanded C GG.CCG triplet repeat sequence.