Substrate preferences of human placental DNA methyltransferase investigated with synthetic polydeoxynucleotides

Abstract

A DNA methyltransferase partly purified from human placenta has been tested on a variety of synthetic polydeoxynucleotides. The results showed that: (i) the enzyme is most active as a ‘maintenance’ or ‘hemi-’ methylase but also has some de novo methylating activity; (ii) the presence or absence of A or T in the substrate strand has little influence on maintenance or de novo activity, while polymers containing C but not G in the same strand are poor de novo substrates and bind poorly to the enzyme; (iii) single-stranded polymers are about as good substrates as double-stranded ones, and the effects of nucleotide composition (particularly G and mC content) on enzyme activity with single strands are similar to those with double-stranded polymers; (iv) strands in which all the cytosines are methylated bind the enzyme well. A mechanism is suggested involving two different sites on the enzyme that recognize CG and mCG, and which rationalizes the activity of eukaryotic DNA methyltransferases towards single-stranded DNA.