DNA Methylation at Mammalian Replication Origins

Abstract

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 ϳ2-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 ϳ500 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.In early embryos undergoing rapid cell cleavage (e.g. frogs, flies, sea urchin, fish), initiation of DNA replication appears neither to require specific DNA sequences nor to occur at specific DNA sites. However, as development progresses past the blastula stage, initiation of DNA replication begins to occur at specific sites (1, 2). Thus, it is not surprising that initiation sites for DNA replication in cultured mammalian cells occur at specific genomic loci (3). For example, a 200-kb 1 region at the human ␤-globin gene (4, 5) and a 500-kb region at the mouse IgH gene (6) are both replicated from a single initiation locus. Moreover, what previously had been viewed as random initiation events distributed throughout "initiation zones" in Schizosaccharomyces pombe and hamster cells more likely reflects the presence of several strongly preferred initiation sites (7,8).Nevertheless, the precise size and composition of these initiation loci, as well as the parameters that define them remain the subject of intense investigation.The fact that specific sites for initiation of DNA replication can be developmentally acquired makes it clear that initiation sites in the metazoa are determined at least in part by epigenetic parameters. These parameters include nuclear structure, chromatin structure, and even...

DNMT1 is a Component of a Multiprotein DNA Replication Complex

“…Another study suggests a slightly longer delay, but showed methylation to be complete by the time the fork had progressed 4 kb. 48,49 DNMT1 co-localizes with PCNA and BrdU to fine nuclear speckles representing early S-phase replication foci and interacts with PCNA in whole cell extracts through a peptide near the amino-terminus (amino acids 163-174) that is distinct from the replication targeting domain described above. 18 The interaction of PCNA with DNMT1 may act to tether DNMT1 to the DNA, thus endowing DNMT1 with processivity by facilitating its tracking along the newly-synthesized DNA.…”

Programming the Transcriptional State of Replicating Methylated DNA

“…DNA methylation at specific cytosine residues might also serve as a signal recognized by the replication machinery, and this may lead to initiation of replication at more specific genomic sites. This mechanism may be used only by a subset of origins because not every origin of replication appears to have methylated CpG sequences (31).…”

Maintenance DNA Methylation of Nucleosome Core Particles

“…Interestingly, DNMT1 also interacts with PCNA at the replication fork (17)(18)(19) and a fraction of CpGs are subject to maintenance methylation prior to maturation of Okazaki fragments on the lagging strand (20,21). This suggests that DNMT1 may need to be present immediately behind the replication fork in order to promote efficient methylation of nascent DNA prior to its packaging into nucleosomes.…”

“…Deposition of newly synthesized histones H3/H4 is mediated, at least in part, by a conserved protein known as chromatin assembly factor 1 (CAF-1), which brings histones H3/H4 to the replication fork via an interaction with the proliferating cell nuclear antigen (PCNA) (12-15). PCNA is a DNA polymerase processivity factor that forms a sliding clamp around DNA and interacts with a number of DNA replication and DNA repair proteins, many of which are constitutively present at the replication fork (16).Interestingly, DNMT1 also interacts with PCNA at the replication fork (17-19) and a fraction of CpGs are subject to maintenance methylation prior to maturation of Okazaki fragments on the lagging strand (20,21). This suggests that DNMT1 may need to be present immediately behind the replication fork in order to promote efficient methylation of nascent DNA prior to its packaging into nucleosomes.…”