Methylation of cytosine residues in DNA plays an important role in regulating 
gene expression during vertebrate embryonic development. Conversely, disruption 
of normal patterns of methylation is common in tumors and occurs early in 
progression of some human cancers. In vertebrates, it appears that the same DNA 
methyltransferase maintains preexisting patterns of methylation during DNA 
replication and carries out de novo methylation to create new methylation 
patterns. There are several indications that inherent signals in DNA structure 
can act in vivo to initiate or block de novo methylation in adjacent DNA 
regions. To identify sequences capable of enhancing de novo methylation of DNA 
in vitro, we designed a series of oligodeoxyribonucleotide substrates with 
substrate cytosine residues in different sequence contexts. We obtained evidence 
that some 5-methylcytosine residues in these single-stranded DNAs can stimulate 
de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase 
as effectively as 5-methylcytosine residues in double-stranded DNA stimulate 
maintenance methylation. This suggests that double-stranded DNA may not be the 
primary natural substrate for de novo methylation and that looped 
single-stranded structures formed during the normal course of DNA replication or 
repair serve as "nucleation" sites for de novo methylation of adjacent DNA 
regions.