Though Dnmt1 is considered the primary maintenance methyltransferase and Dnmt3a and Dnmt3b are considered de novo methyltransferases in mammals, these three enzymes may work together in maintaining as well as establishing DNA methylation patterns. It has been proposed that Dnmt1 may carry out de novo methylation at sites in the genome with transient single-stranded regions, such as replication origins, and then spread methylation from these nucleation sites in vivo, even though such activity has not been reported.

In this study, we show that Dnmt3a does not act on single-stranded substrates in vitro, indicating that Dnmt3a is not likely to initiate DNA methylation at such proposed nucleation sites. Dnmt3a shows similar methylation activity on unmethylated and hemimethylated duplex DNA, though with some substrate preference. Unlike Dnmt1, pre-existing cytosine methylation at CpG sites or non-CpG sites does not stimulate Dnmt3a activity in vitro and in vivo.

The fact that Dnmt3a does not act on single stranded DNA and is not stimulated by pre-existing cytosine methylation indicates that the de novo methylation activity of Dnmt3a is quite different from that of Dnmt1. These findings are consistent with a model in which Dnmt3a initiates methylation on one of the DNA strands of duplex DNA, and these hemimethylated sites then stimulate Dnmt1 activity for further methylation.