Identifying a property of origins of DNA synthesis required to support plasmids stably in human cells

Abstract

The plasmid origin of replication, oriP, of Epstein-Barr Virus (EBV) was identified in an assay to detect autonomously replicating sequences (ARSs) in human cells. Raji ori, a second origin in EBV, functions in vivo but fails in long-term ARS assays. We examined the initiating element, DS, within oriP and Raji ori to resolve this paradox. DS, but not Raji ori, binds EBNA1; whereas both act as ARSs in short-term assays, with DS being more efficient, only DS can act as an ARS in long-term assays. Surprisingly, we found that DS supported the establishment of a plasmid with Raji ori in cis and that after deletion of DS, Raji ori could now act as an ARS in the long term. This finding explains the frequent failure of ARS assays in mammalian cells. More origins can initially act as ARSs than can be established. We identified one requirement for ARSs to be established: They must function efficiently enough initially to generate a wide distribution of numbers of plasmids per cell. Only the cells that have more than a threshold number of plasmids can survive selections imposed on the cells to retain these replicons.autonomously replicating sequence ͉ DNA replication ͉ Epstein-Barr virus T he study of DNA replication in the budding yeast Saccharomyces cerevisiae has provided much of the foundation of our understanding of DNA replication in eukaryotic cells. In S. cerevisiae, assays for autonomously replicating sequences (ARS) were used to identify the origins of DNA synthesis after their introduction into plasmids having a functional centromere (CEN) (1-5). Unlike ARSs in yeast, origins of DNA synthesis in mammalian cells are poorly understood. Parallel experiments in mammalian cells have not been the standard initially used to identify chromosomal origins of DNA synthesis. One difficulty in developing a mammalian ARS assay, for example, is imposed by the size of mammalian centromeres making their use difficult.One mammalian origin of DNA synthesis that was identified in an ARS assay in human cells is the Dyad symmetry (DS) of Epstein-Barr virus (EBV) (6, 7). DS is a discrete, licensed origin that functions in a variety of mammalian hosts in the presence of the viral trans-acting element EBNA1 (Epstein-Barr nuclear antigen 1) (8). It requires the family of repeats (FR) from EBV in cis and EBNA1 in trans to provide its segregation mechanism to the replicon (9-11). FR and EBNA1 thus functionally replace the requirement in yeast for a CEN element in ARS assays. A second origin of DNA synthesis has been identified in the EBV genome, which also functions extrachromosomally and is not discrete but does support licensed DNA synthesis (Fig. 1A) (12-15). These latter properties make this origin, which we term ''Raji ori,'' similar to well studied mammalian chromosomal origins such as the DHFR (16) and ␤-globin (17, 18). Raji ori was identified by two-dimensional gel electrophoresis and in an assay termed ''single molecule analysis of replicated DNA'' (SMARD) and was found both to be the predominant origin of DNA synthesis used in t...

Identifying Sites Bound by Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) in the Human Genome: Defining a Position-Weighted Matrix To Predict Sites Bound by EBNA1 in Viral Genomes

“…Aligned below A and B are the regions (approximately 500 to 700 bp) from the RRAS/SR-A1 promoter or the SELK/ACTR8 promoter used in C for EMSA analysis. A DNA fragment containing the two Rep* sites was used as a positive control, yielding two shifted signals in the presence of EBNA1-DBD; a DNA fragment from Raji ori was used as a negative control (50). The DNA fragments are of various sizes, so the unbound DNAs do not move similarly in the gel.…”

“…(B) An EMSA was performed using PCR-amplified DNA fragments corresponding to specific regions in EBV (DNA fragments were between 400 and 500 bp in length). A DNA fragment containing the two Rep* sites was used as a positive control, yielding two shifted signals in the presence of EBNA1-DBD; a DNA fragment from Raji ori was used as a negative control (50). The DNA fragments are of various sizes, so the unbound DNAs do not move similarly in the gel.…”

Replication of Epstein-Barr Viral DNA

“…The efficient initial replication of replicons with DS plus FR allows them to achieve this distribution needed to be established. Replicons with Raji ori and FR fail to be established but can be maintained in Raji cells once established by virtue of having had DS in cis (Wang and Sugden 2008). Thus, the DS replicator is peculiarly efficient on being introduced into cells, a function required during virus infection to allow EBV to be established.…”

“…EBNA1 does not contribute directly to origin function of Raji ori, though. EBNA1 does not bind detectably to Raji ori as measured by gel shift assays using 40 overlapping fragments of 600 bp that span this zone of initiation (Wang and Sugden 2008). Given that Raji ori is a zone with multiple sites for the initiation of licensed DNA replication, ORC likely binds those sites but the means by which it does so is unknown.…”

“…The DNA replication mediated by DS at oriP is more efficient than that of Raji ori with FR in cis and EBNA1 in trans when initially introduced into Raji cells. This more efficient replication allows plasmids with DS plus FR to become "established" (Wang and Sugden 2008). Only 1% -10% of EBV's plasmid replicons that are introduced into cells and initially replicate progress to be maintained stably after 15 generations or so, a process termed "establishment" (Leight and Sugden 2001).…”