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J Biol Chem, Vol. 274, Issue 36, 25792-25800, September 3, 1999

DNA Methylation at Mammalian Replication Origins

Theo Rein, Takehiko Kobayashi^¶, Michelle Malott, Michael Leffak, and Melvin L. DePamphilis

From the NICHD, National Institutes of Health, Bethesda, Maryland 20892-2753, the  Max Planck Institut für Psychiatrie, Kraepelinstrasse 2-10, D-80804 München, Germany, the ^¶ National Institute for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki 444, Japan, and the  Department of Biochemistry and Molecular Biology, Wright State University, Dayton, Ohio 45435

In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the ~2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin  (ori-), downstream of the hamster DHFR gene. Remethylation at ori- did not begin until ~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 regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori- activity. Cells that were >50% reduced in methylation at ori- no longer selectively activated ori-. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.

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