Abstract
In mammalian cells, the activity of the sites of initiation of DNA replication appears to be influenced epigenetically, but this regulation is not fully understood. Most studies of DNA replication have focused on the activity of individual initiation sites, making it difficult to evaluate the impact of changes in initiation activity on the replication of entire genomic loci. Here, we used single molecule analysis of replicated DNA (SMARD) to study the latent duplication of Epstein-Barr virus (EBV) episomes in human cell lines. We found that initiation sites are present throughout the EBV genome and that their utilization is not conserved in different EBV strains. In addition, SMARD shows that modifications in the utilization of multiple initiation sites occur across large genomic regions (tens of kilobases in size). These observations indicate that individual initiation sites play a limited role in determining the replication dynamics of the EBV genome. Long-range mechanisms and the genomic context appear to play much more important roles, affecting the frequency of utilization and the order of activation of multiple initiation sites. Finally, these results confirm that initiation sites are extremely redundant elements of the EBV genome. We propose that these conclusions also apply to mammalian chromosomes.
Highlights
Biochemical studies performed in higher eukaryotes have shown that DNA replication initiates at specific sites, or within initiation zones, suggesting the involvement of particular DNA sequences called replicators
This could explain why only weak bubble arcs were detected by 2D gel electrophoresis throughout the region that usually replicates first (RRF), even though the duplication of the episomes usually begins within this genomic region
We conclude that the RRFs in the Raji and Mutu I Epstein-Barr virus (EBV) genomes are similar in that they contain various initiation sites that have a shared tendency to be activated at the beginning of the duplication of each episome
Summary
Biochemical studies performed in higher eukaryotes have shown that DNA replication initiates at specific sites, or within initiation zones, suggesting the involvement of particular DNA sequences called replicators (reviewed by DePamphilis 1999). Initiation of DNA replication at oriP was recently shown to be regulated by geminin, and to correlate with the binding of various cellular components of the replication complex (Orc, Orc, Orc, Orc, Orc, Mcm, Mcm, and Mcm7) (Chaudhuri et al 2001; Dhar et al 2001; Schepers et al 2001; Ritzi et al 2003). These and other reports have been interpreted as evidence that oriP contains a replicator (e.g., Koons et al 2001). Reports from different laboratories have shown that various portions of the EBV genome, including oriP, can be deleted without affecting the maintenance of the episomes in replicating cells
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