Interaction between DNMT1 and DNA replication reactions in the SV40 in vitro replication system.

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.