We have shown that in several mammalian cell lines a minor fraction of cytosine methylation is delayed for up to several hours after strand synthesis and that different methylases performed the immediate and the delayed classes of DNA methylation. To investigate the time course of this delayed methylation we have used three different cell lines, two of human and one of hamster origin. These were synchronized by two different methods: mitotic detachment and double hydroxyurea blocks. A uniform picture was obtained with all three cell lines. Delayed methylation of early replicating sequences occurred while cells were still in mid-S-phase, with the maximum rate of delayed methylation occurring in cells in the second half of S and in G2. Delayed methylation seems to be complete before cells entered the next G1-phase. Limited DNAase I hydrolysis of cell nuclei was used to test whether the delay in methylation in some DNA sequences was due to high levels of transcriptional activity. However, DNA sequences exhibiting delayed methylation showed no preferential concentration in or exclusion from DNAase I hypersensitive regions.
Animals Cell Line