BACKGROUND: 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.
RESULTS: 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.
CONCLUSION: 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.