In contrast to normal cells, cancer cells exhibit both genetic and epigenetic 
instability. These unique properties give rise to genetic and epigenetic 
heterogeneity in a given population of cancer cells and provide a means for the 
population to undergo phenotypic progression by clonal selection. DNA 
methylation at CpG dinucleotides is one of the epigenetic marks that are 
frequently disturbed in cancer cells. To understand how the CpG methylation 
pattern is changeable in cancer cells, it is necessary to know how it is 
faithfully maintained in normal cell proliferation. Toward this goal, we have 
developed a novel in vitro system that is based on the well-established SV40 in 
vitro replication system and functions to reconstitute concurrent DNA 
replication and DNA maintenance methylation reactions. We found that DNA 
methylation was maintained only when exogenous DNA methyltransferase 1 (DNMT1) 
and S-adenosyl methionine (SAM) were added to the reaction. We demonstrated that 
DNMT1 associates with replicating and/or replicated chromatin irrespective of 
the DNA methylation status of template DNA. Moreover, the PCNA-binding domain 
(PBD) of DNMT1 is not required for the association. Taken together, we suggest 
that DNMT1 is recruited to replicating and/or replicated chromatin in a 
constitutive manner independent of the DNA methylation reaction. The in vitro 
system described in this report is very useful for analyzing the molecular 
mechanism underlying the DNA maintenance methylation reaction.