Fig 1.
Genetic maps of the minichromosomes used in this study.
On top pAC_SV40ori (6,126 bp) that contains the SV40 bi-directional origin of replication (outlined in red) showing its most relevant elements. In the middle pAC_EBVoriP (6,108 bp) that contains the EBV bidirectional origin of replication (oriP), including the “dyad symmetry” element (DS) and the “family of repeats” (FR). At the bottom, pEco3’Δ (6,377 bp) containing the SV40 bi-directional origin of replication (in red) and the EBV’s FR (outlined in blue and yellow).
Fig 2.
Intact forms of pAC_SV40ori isolated from E. coli and HEK 293T cells analyzed by two-dimensional agarose gel electrophoresis (2D gels) where the first dimension occurred without or in the presence of different concentrations of chloroquine.
The electrophoretic mobility of topoisomers with ΔLk = 0 during the first and second dimensions are indicated by horizontal and perpendicular broken red lines in the panel run with 0.50 μg/ml chloroquine. The blue arrow points the transition to a non-B conformation in the 2D gel where the first dimension occurred in the presence of 1.00 μg/ml chloroquine.
Fig 3.
Intact forms of pAC_EBVoriP isolated from E. coli and HEK 293E cells analyzed by two-dimensional agarose gel electrophoresis (2D gels) where the first dimension occurred without or in the presence of different concentrations of chloroquine.
The electrophoretic mobility of topoisomers with ΔLk = 0 during the first and second dimensions are indicated by horizontal and perpendicular broken red lines in the panel run with 0.50 μg/ml chloroquine. The blue arrow points the transition to a non-B conformation in the 2D gel where the first dimension occurred in the presence of 1.00 μg/ml chloroquine.
Fig 4.
Cartoons describing in a diagrammatic manner the behavior of (-) and (+) supercoiled topoisomers in 2D gels without or where the first dimension occurred in the presence of different concentrations of an intercalator.
We consider a theoretical population of 13 topoisomers with a ΔLk ranging from +2 to -10 (indicated as such in all diagrams), with the modal topoisomer being ΔLk = -5. The diameter of the circles indicates their abundance in the population. To simplify the model, we established that in these 2D gels resolution would go from ΔLk = 0 to ΔLk = +/- 8 (in real 2D gels resolution goes from ΔLk = 0 to ΔLk = ~ +/- 20). Diagrams to the far left show the electrophoretic mobility of these topoisomers after the first (top) and second (bottom) dimensions run in the absence of intercalating drugs. Vertical broken lines highlight the mobility of each topoisomer after the first and second dimensions. Second left diagrams show their electrophoretic mobility when the first dimension occurred in the presence of an intercalator that introduced four (+) turns to every topoisomer. Diagrams second to right show their electrophoretic mobility when the first dimension occurred in the presence of an intercalator that introduced six (+) turns to every topoisomer. Finally, far right diagrams show their electrophoretic mobility when the first dimension occurred in the presence of an intercalator that introduced eight (+) turns to every topoisomer. Each filled circle represents individual topoisomers. An empty circle corresponds nicked circles. The half black circle represents a topoisomer that migrated as heavily supercoiled (CCC) during the first dimension, was nicked between the first and second dimensions and migrated as a relaxed molecule (OC) during the second dimension. Color code: black circles represent topoisomers that migrated as (-) supercoiled during the first and second dimensions in the absence of chloroquine. Green circles represent those topoisomers that turned (+) supercoiled in the presence of the intercalator during the first dimension. Red circles represent those topoisomers that remained (-) supercoiled in the presence of the intercalator during the first dimension. Note that their number diminished as the (+) ΔLk introduced by the intercalator increased. Violet circles represent those topoisomers that remained (+) supercoiled during the first and second dimensions. Note that after the second dimension run, highly (-) supercoiled topoisomers appear at the bottom whereas (+) supercoiled topoisomers appear at the top right.
Fig 5.
Histogram showing the efficiency of different concentrations of chloroquine to introduce (+) ΔLk in pAC_SV40ori (6,126 bp) and pAC_EBVoriP (6,108 bp).
The number of (+) ΔLk introduced by the different concentrations of chloroquine corresponds to the number of topoisomers that lay between the topoisomer that migrated with a ΔLk = 0 after the first dimension and the topoisomer that migrated with a ΔLk = 0 after the second dimension (see broken red lines in Figs 2 and 3). Note that the net number of (+) supercoils introduced was almost identical in both cases.
Fig 6.
Intact forms of pEco3’Δ isolated from HEK 293T cells in the absence or presence of EBNA-1 analyzed by two-dimensional agarose gel electrophoresis (2D gels) and their corresponding Western blots.
(A) Note that topoisomers with (-) and (+) supercoiling were clearly distinguished. The panel shown on top corresponds to DNA isolated from cells without EBNA-1. Those shown below were isolated from cells in the presence of EBNA-1. Panels to the left were analyzed in 2D gels run without chloroquine whereas those to the right were analyzed in 2D gels where the first dimension occurred in the presence of 1.00 μg/ml chloroquine. The blue arrows point the transition to a non-B DNA conformation. (B) Total cellular proteins from HEK 293T cells co-transfected with pEco3’Δ and pCXWB-EBNA1 (column 1) and from the same cell line transfected with pEco3’Δ alone (column 2) subjected to Western blotting for EBNA-1 and α-Tubulin.
Fig 7.
Most of pAC_EBVoriP DNA transfected into HEK 293E cells remained unreplicated up to 40 hours after transfection.
pAC_EBVoriP DNA isolated from E. coli and from HEK 293E cells 40 hours after transfection was analyzed intact and after digestion with DpnI. Selected non-radioactive hybridization (A) and ethidium bromide staining (B) showed that in both cases DpnI digested almost all the DNA. In addition, DNA of the minichromosomes isolated from HEK 293 cells (that do not express EBNA-1) were analyzed in 2D gels run without chloroquine (to the left) and in 2D gels where the first dimension occurred in the presence of 1.00 μg/ml chloroquine (C). As these minichromosomes were isolated from HEK 293 cells that do not express EBNA-1, they cannot possibly support oriP initiation of replication. The blue arrows point the transition to a non-B DNA conformation.
Fig 8.
Schematic model illustrating the interaction of EBNA-1 with the DS and the FR of oriP in two steps.
A: In the first step EBNA-1 binds to its cognate DNA sites wrapped around nucleosomes throughout most of the cell cycle. This interaction does not cause removal or the precise positioning of nucleosomes within or adjacent to the FR and DS elements of oriP. B: In the second step, which is cell cycle specific, an unknown factor (?) causes structural conformation changes that leads to the formation of a loop between the DS and the FR. This conformational change allows the melting of A+T rich regions and initiation of DNA replication close to the DS element. C: Once proteins are removed the minichromosome shows a linking difference (ΔLk) corresponding to approximately -1 per nucleosome removed. The gray circle highlights a RH crossing. In the diagram the original molecule shown in (A) would end-up negatively supercoiled with a ΔLk = -12. The relative position of the DS and the FR are shown in yellow and blue and the putative replication initiation site is marked in red. Double-stranded DNA is represented in green and blue.