DNA methylation at mammalian replication origins

In Escherichia coli, DNA methylation regulates both origin usage and the ti me required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high d ensity cluster of methylated CpG dinucleotides, and others whose methylatio n status has not yet been characterized have the potential to exhibit a sim ilar DNA methylation pattern. One of these origins is found within the simi lar to 2-kilobase pair region upstream of the human c-myc gene that contain s 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosi nes at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methyl ation is not a universal component of mammalian replication origins, To det ermine whether or not DNA methylation plays a role in regulating the activi ty of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin beta (ori-beta), downstream o f the hamster DHFR gene. Remethylation at ori-beta did not begin until simi lar to 500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in reg ulating reassembly of prereplication complexes in mammalian cells, as it do es in E. coli, To determine whether or not DNA methylation plays any role i n origin activity, hypomethylated hamster cells were examined for ori-beta activity. Cells that were >50% reduced in methylation at ori-beta no longer selectively activated ori-beta. Therefore, at some loci, DNA methylation e ither directly or indirectly determines where replication beans.