Supercoiling, knotting and replication fork reversal in partially replicated plasmids

Abstract

To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive DeltaLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide.

Figure 1

Maps of the plasmids used in this study. (A) Map of pBR18-TerE@StyI showing the relative position of its most relevant features: the ColE1 unidirectional origin, the E.coli terminator TerE, the ampicillin resistance and rop genes and the recognition sites for a number of restriction endonucleases that cut the plasmid only once. (B) TerE binds the terminator protein TUS and the Ter–TUS complex acts as a polar replication fork barrier. Therefore, blockage of the replication fork at TerE would lead to the accumulation of a specific RI containing an internal bubble with a total mass 1.26 times the mass of non-replicating plasmids. (C) Map of pBR18-TerE@EcoRI showing the position of its most relevant features. (D) In this plasmid, blockage of the replication fork at TerE would lead to the accumulation of a specific RI containing an internal bubble and with a total mass 1.57 times the mass of non-replicating plasmids.

Figure 2

Autoradiograms of 2D gels corresponding to pBR18-TerE@StyI (A) and pBR18-TerE@EcoRI (C) after digestion with ScaI and their corresponding diagrammatic interpretations (B and D). Note that the number and complexity of knotted bubbles was significantly higher in pBR18-TerE@EcoRI.

Figure 3

2D gels and electron microscopy confirmed that in vivo partially replicated forms of pBR18-TerE@StyI and pBR18-TerE@EcoRI accumulated with the predicted masses. (A) 2D gel of intact forms of pBR18-TerE@StyI. (B) Diagrammatic interpretation of the different signals observed in the autoradiogram shown in (A). (C) Electron microscopy of a selected molecule that was eluted from the signal circled in (A). (D) Diagrammatic interpretation of the electron micrograph shown in (C). The black line represents unreplicated DNA while the stippled lines represent replicated arms. Numbers denote the percentage contour length of unreplicated and replicated arms, respectively. (E) 2D gel of intact forms of pBR18-TerE@EcoRI. (F) Diagrammatic interpretation of the different signals observed in the autoradiogram shown in (E). (G) Electron micrograph of a selected molecule that was eluted from the signal circled in (E). (H) Diagrammatic interpretation of the electron micrograph shown in (G). As in the previous case, the black line represents unreplicated DNA while the stippled lines represent replicated arms. Numbers denote the percentage contour length of unreplicated and replicated arms. CCC, covalently closed circle; OC, open circle; RIs, replication intermediates; σRIs, broken replication intermediates; L, linears.

Figure 4

Autoradiograms of 2D gels corresponding to pBR18-TerE@StyI (upper) and pBR18-TerE@EcoRI (lower) where the second dimension occurred without (left) or in the presence of different concentrations of chloroquine (concentrations in µg/ml are indicated at the top). All the autoradiograms were aligned so that the positions of the OC and OCRIs, which are not affected by drug concentration, coincided. The positions of the CCC and CCCRIs are indicated only in the autoradiograms corresponding to untreated panels (left).

Figure 5

Autoradiograms of 2D gels corresponding to pBR18-TerE@StyI (upper) and pBR18-TerE@EcoRI (lower) where the second dimension occurred without (left) or in the presence of different concentrations of ethidium bromide (concentrations in µg/ml are indicated at the top). All the autoradiograms were aligned so that the positions of the OC and OCRIs, which are not affected by drug concentration, coincided. The positions of the CCC and CCCRIs are indicated only in the autoradiograms corresponding to untreated panels (left).

Figure 6

Diagrammatic model illustrating how the ΔLk value of a DNA segment containing a stalled fork might change as the concentration of chloroquine or ethidium bromide increases. At low concentrations of the drug the native negative supercoiling would progressively diminish until the molecule becomes completely relaxed (ΔLk = 0). From this point on, increasing concentrations of the drug would induce a net positive supercoiling. However, as soon as the ΔLk value becomes positive, nascent strands would separate from their corresponding parental strands and re-anneal with each other, leading to fork reversal. Each positive ΔLk eliminated would force fork reversal to advance 10 bp [adapted from Alexandrov et al. (31)].

Figure 7

Electron micrographs of intact forms of pBR18-TerE@StyI showing reversed forks. CCCRIs were eluted from agarose gels in the presence of 100 µg/ml chloroquine, following which the drug was removed by washing thoroughly with distilled water and the CCCRIs treated with topoisomerase I to eliminate all negative supercoiling, exposed to 100 µg/ml chloroquine again and examined under an electron microscope. Black lines represent unreplicated DNA, stippled lines replicated arms and horizontally striped lines the newly formed nascent–nascent fourth arm that resulted from fork reversal (indicated by arrows). Numbers denote the percentage contour length of unreplicated and replicated arms.

Figure 8

Diagram of the structure of a replication bubble where initiation occurred at the ColE1 origin and the replication fork stopped at the Ter–TUS complex. Thin lines represent parental DNA while thick lines represent nascent DNA. The two vertical dashed lines mark the position of the origin and the proximal end of the TUS recognition sequence, respectively. Numbers indicate base pairs before (–) and after (+) the origin and the right end of the TUS recognition sequence.