CONCURRENT REPLICATION AND METHYLATION AT MAMMALIAN ORIGINS OF REPLICATION

Observations made with Escherichia coli have suggested that a lag betw een replication and methylation regulates initiation of replication. T o address the question of whether a similar mechanism operates in mamm alian cells, we have determined the temporal relationship between init iation of replication and methylation in mammalian cells both at a com prehensive level and at specific sites. First, newly synthesized DNA c ontaining origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, Africa n green monkey kidney fibroblasts [CV-1], and primary African green mo nkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysi s, the levels of cytosine methylation residing in all four possible di nucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomi c DNAs were equivalent, suggesting that DNA replication and methylatio n are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA, Ho wever, in contrast to previous observations, neither nascent nor genom ic DNA contained detectable levels of methylated cytosines at dinucleo tide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylat ed). The nearest-neighbor analysis also shows that cancer cell lines a re hypermethylated in both nascent and genomic DNAs relative to the pr imary cell lines. The extent of methylation in nascent and genomic DNA s at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and beta-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with re plication. Interestingly, the c-myc origin was found to be unmethylate d in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nucl ear antigen in the replication fork.