The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells

Nanbo, Asuka; Sugden, Arthur U.; Sugden, Bill

doi:10.1038/sj.emboj.7601853

Cited by 171 publications

(207 citation statements)

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“…Consistent with the involvement of Epstein-Barr Virus (EBV) in NPC etiology and previous estimates of 1-30 viral genome copies per cell (Nanbo et al 2007;Liu et al 2011), we detected large amounts of EBV genomic sequence in NPC-5989 but not in its matched normal sample (1.86 million versus 521 read pairs). We estimate that there were about 108 EBV genome copies per cell in the tumor sample (Supplemental Material).…”

Section: Resultssupporting

confidence: 88%

Discovery of recurrent structural variants in nasopharyngeal carcinoma

et al. 2013

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We present the discovery of genes recurrently involved in structural variation in nasopharyngeal carcinoma (NPC) and the identification of a novel type of somatic structural variant. We identified the variants with high complexity mate-pair libraries and a novel computational algorithm specifically designed for tumor-normal comparisons, SMASH. SMASH combines signals from split reads and mate-pair discordance to detect somatic structural variants. We demonstrate a >90% validation rate and a breakpoint reconstruction accuracy of 3 bp by Sanger sequencing. Our approach identified three in-frame gene fusions (YAP1-MAML2, PTPLB-RSRC1, and SP3-PTK2) that had strong levels of expression in corresponding NPC tissues. We found two cases of a novel type of structural variant, which we call ''coupled inversion,'' one of which produced the YAP1-MAML2 fusion. To investigate whether the identified fusion genes are recurrent, we performed fluorescent in situ hybridization (FISH) to screen 196 independent NPC cases. We observed recurrent rearrangements of MAML2 (three cases), PTK2 (six cases), and SP3 (two cases), corresponding to a combined rate of structural variation recurrence of 6% among tested NPC tissues.

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Section: Resultssupporting

confidence: 88%

Discovery of recurrent structural variants in nasopharyngeal carcinoma

et al. 2013

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“…Our recent analysis of EBV plasmids in live cells found that Ϸ16% of these DNAs fail to be synthesized each S phase (11). EBV compensates for this failure by providing its host cell a selective advantage and by errors in its partitioning that generate cells with a wide distribution of numbers of plasmids per cell.…”

Section: Ds But Not Raji Ori Can Generate a Broad Distribution Of Nmentioning

confidence: 99%

“…The efficiencies of Raji ori and Raji middle to act as stable ARSs were then measured in two ways: (i) Cells were cultured in the absence of selection for increasing times and subsequently plated into selective media in 96-well plates to measure their colony-forming ability. In this experiment, only the efficiency with which the various origins support the initiation of DNA synthesis determines the rate of loss of plasmids from the cells (11). The more efficient the origin is, the more slowly the cells lose the drug-resistant plasmid and the more efficiently colonies arise after selection is reapplied.…”

Section: Raji Ori's and Raji Middle's Functioning As Arss After Estabmentioning

confidence: 99%

“…The initial efficiency of DNA synthesis of DS allows the evolution over 10-20 generations of a population of daughter cells with a wide distribution in numbers of plasmids per cell. Such a wide distribution is required to sustain cells' resistance to selection (11). ARS assays thus require peculiarly efficient origins of DNA synthesis in mammalian cells.…”

mentioning

confidence: 99%

“…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)(10)(11). FR and EBNA1 thus functionally replace the requirement in yeast for a CEN element in ARS assays.…”

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

Wang

Sugden

2008

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Episomal Vectors

2011

Primary and Stem Cells

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The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells

Cited by 171 publications

References 39 publications

Discovery of recurrent structural variants in nasopharyngeal carcinoma

Discovery of recurrent structural variants in nasopharyngeal carcinoma

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

Episomal Vectors

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