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Mol Cell Biol, June 1998, p. 3475-3482, Vol. 18, No. 6
McGill Cancer Centre,1
Department of Biochemistry,2 and
Department of Pharmacology and
Therapeutics,3 McGill University, Montreal,
Quebec, Canada H3G 1Y6
Received 30 June 1997/Returned for modification 16 December
1997/Accepted 12 March 1998
Observations made with Escherichia coli have suggested
that a lag between replication and methylation regulates
initiation of replication. To address the question of whether a similar
mechanism operates in mammalian cells, we have determined the temporal
relationship between initiation of replication and methylation in
mammalian cells both at a comprehensive level and at specific sites.
First, newly synthesized DNA containing origins of replication was
isolated from primate-transformed and primary cell lines (HeLa cells,
primary human fibroblasts, African green monkey kidney
fibroblasts [CV-1], and primary African green monkey kidney cells) by
the nascent-strand extrusion method followed by sucrose gradient
sedimentation. By a modified nearest-neighbor analysis, the
levels of cytosine methylation residing in all four possible
dinucleotide sequences of both nascent and genomic DNAs were
determined. The levels of cytosine methylation observed in the nascent
and genomic DNAs were equivalent, suggesting that DNA replication and
methylation 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. However, in contrast to previous observations,
neither nascent nor genomic DNA contained detectable levels of
methylated cytosines at dinucleotide contexts other than CpG (i.e.,
CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis
also shows that cancer cell lines are hypermethylated in both nascent
and genomic DNAs relative to the primary cell lines. The extent of
methylation in nascent and genomic DNAs at specific sites was
determined as well by bisulfite mapping of CpG sites at the lamin B2,
c-myc, and
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Copyright © 1998, American Society for Microbiology. All rights reserved.
Concurrent Replication and Methylation at Mammalian
Origins of Replication
-globin origins of replication. The
methylation patterns of genomic and nascent clones are
the same, confirming the hypothesis that methylation occurs
concurrently with replication. Interestingly, the c-myc
origin was found to be unmethylated 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 nuclear antigen in the
replication fork.
*
Corresponding author. Mailing address: Department
of Pharmacology, McGill University, 3655 Drummond St., Montreal, PQ
H3G1Y6 Canada. Phone: (514) 398-7107. Fax: (514) 398-6690. E-mail: M52YF{at}Pharma.McGill.CA.
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