Plasmid DNA replication and topology as visualized by two-dimensional agarose gel electrophoresis.

During the last 20 years, two-dimensional agarose gel electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of uni-directional replication, replication origin interference, DNA breakage at the forks, replication fork blockage, replication knotting, replication fork reversal, the interplay of supercoiling and catenation and other changes in DNA topology that take place as replication progresses.

A redundancy of processes that cause replication fork stalling enhances recombination at two distinct sites in yeast rDNA.

DNA recombination was investigated by monitoring integration at the rDNA of a circular minichromosome containing a 35S minigene and a replication fork barrier (RFB). The effects of replication fork stalling on integration were studied in wild-type, FOB1Delta, SIR2Delta and the double mutant FOB1DeltaSIR2Delta cells. The results obtained confirmed that Sir2p represses and replication fork stalling enhances integration of the minichromosome. This integration, however, only took place at two distinct sites: the RFB and the 3' end of the 35S gene. For integration to take place at the 35S gene, replication fork stalling must occur at the 3' end of the gene in both the minichromosome and the chromosomal repeats. Integration at the RFB, on the other hand, occurred readily in FOB1Delta cells, indicating that more than a single mechanism triggers homologous recombination at this site. Altogether, these observations strongly suggest that the main role for replication fork stalling at the rDNA locus is to promote homologous recombination rather than just to prevent head-on collision of transcription and replication as originally thought.

Knotting dynamics during DNA replication.

The topology of plasmid DNA changes continuously as replication progresses. But the dynamics of the process remains to be fully understood. Knotted bubbles form when topo IV knots the daughter duplexes behind the fork in response to their degree of intertwining. Here, we show that knotted bubbles can form during unimpaired DNA replication, but they become more evident in partially replicated intermediates containing a stalled fork. To learn more about the dynamics of knot formation as replication advances, we used two-dimensional agarose gel electrophoresis to identify knotted bubbles in partially replicated molecules in which the replication fork stalled at different stages of the process. The number and complexity of knotted bubbles rose as a function of bubble size, suggesting that knotting is affected by both precatenane density and bubble size.

Supercoiling, knotting and replication fork reversal in partially replicated plasmids.

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.

Premature termination of DNA replication in plasmids carrying two inversely oriented ColE1 origins.

In Escherichia coli plasmids carrying two inversely oriented ColE1 origins, DNA replication initiates at only one of the two potential origins. The other silent origin acts as a replication fork barrier. Whether this barrier is permanent or simply a pausing site remains unknown. Here, we used a repeated primer extension assay to map in vivo, at the nucleotide level, the 5' end of the nascent strand where initiation and blockage of replication forks occurs. Initiation occurred primarily at the previously defined origin, however, an alternative initiation site was detected 17 bp upstream. At the barrier, the lagging strand also terminated at the main initiation site. Therefore, the 5' end of the nascent strand at the barrier was identical to that generated during initiation. This observation strongly suggests that blockage of the replication fork at the silent origin is not just a pausing site but permanent, and leads to a premature termination event.

Bi-directional replication and random termination.

Two-dimensional (2D) agarose gel electrophoresis was used to study termination of DNA replication in a shuttle vector, YRp7', when it replicated in Escherichia coli, Saccharomyces cerevisiae and Xenopus egg extracts. In E. coli, the 2D gel patterns obtained were consistent with uni-directional replication initiated at a specific site, the ColE1 origin. In consequence, termination also occurred precisely at the ColE1 origin. In Xenopus egg extracts, the particular shape of the bubble arc as well as the triangular smear detected to the left of the simple-Y pattern indicated random initiation and termination. In S.cerevisiae, initiation occurred at the ARS1 origin and replication proceeded in a bi-directional manner. However, termination did not always occur at a specific site 180 degrees across from the origin, but almost all along the south hemisphere of the plasmid. Inversion, deletion or replacement of DNA sequences located throughout this hemisphere did not eliminate random termination. Analysis of the replication intermediates of another yeast plasmid bearing a different origin, ARS305, also exhibited random termination. We propose that the random termination events observed in S.cerevisiae could be due to an asynchronous departure of both forks from the bi-directional origin in addition to differences in the rate of fork progression. These observations could be extended to all bi-directional origins.

Formation of knots in partially replicated DNA molecules.

Bacterial plasmids with two origins of replication in convergent orientation are frequently knotted in vivo. The knots formed are localised within the newly replicated DNA regions. Here, we analyse DNA knots tied within replication bubbles of such plasmids, and observe that the knots formed show predominantly positive signs of crossings. We propose that helical winding of replication bubbles in vivo leads to topoisomerase-mediated formation of knots on partially replicated DNA molecules.

DnaB helicase is unable to dissociate RNA-DNA hybrids. Its implication in the polar pausing of replication forks at ColE1 origins.

A series of plasmids were constructed containing two unidirectional ColE1 replication origins in either the same or opposite orientations and their replication mode was investigated using two-dimensional agarose gel electrophoresis. The results obtained showed that, in these plasmids, initiation of DNA replication occurred at only one of the two potential origins per replication round regardless of origins orientation. In those plasmids with inversely oriented origins, the silent origin act as a polar pausing site for the replication fork initiated at the other origin. The distance between origins (up to 5.8 kilobase pairs) affected neither the interference between them to initiate replication nor the pausing function of the silent origin. A deletion analysis indicated that the presence of a transcription promoter upstream of the origin was the only essential requirement for it to initiate replication as well as to account for its polar pausing function. Finally, in vitro helicase assays showed that Escherichia coli DnaB is able to melt DNA-DNA homoduplexes but is very inefficient to unwind RNA-DNA hybrids. Altogether, these observations strongly suggest that replication forks pause at silent ColE1 origins due to the inability of DnaB helicase, which leads the replication fork in vivo, to unwind RNA-DNA hybrids.

Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis.

Neutral/neutral two-dimensional (2D) agarose gelelectrophoresis was used to investigate populations of the different topological conformations that pBR322 can adopt in vivo in bacterial cells as well as in Xenopus egg extracts. To help in interpretation and identification of all the different signals, undigested as well as DNA samples pretreated with DNase I, topoisomerase I and topoisomerase II were analyzed. The second dimension of the 2D gel system was run with or without ethidium bromide to account for any possible changes in the migration behavior of DNA molecules caused by intercalation of this planar agent. Finally, DNA samples were isolated from a recA-strain of Escherichia coli , as well as after direct labeling of the replication intermediates in extracts of Xenopus laevis eggs. Altogether, the results obtained demonstrated that 2D gels can be readily used to identify most of the complex topological populations that circular molecules can adopt in vivo in both bacteria and eukaryotic cells.

The migration behaviour of DNA replicative intermediates containing an internal bubble analyzed by two-dimensional agarose gel electrophoresis.

Initiation of DNA replication in higher eukaryotes is still a matter of controversy. Some evidence suggests it occurs at specific sites. Data obtained using two-dimensional (2D) agarose gel electrophoresis, however, led to the notion that it may occur at random in broad zones. This hypothesis is primarily based on the observation that several contiguous DNA fragments generate a mixture of the so-called 'bubble' and 'simple Y' patterns in Neutral/neutral 2D gels. The interpretation that this mixture of hybridisation patterns is indicative for random initiation of DNA synthesis relies on the assumption that replicative intermediates (RIs) containing an internal bubble where initiation occurred at different relative positions, generate comigrating signals. The latter, however, is still to be proven. We investigated this problem by analysing together, in the same 2D gel, populations of pBR322 RIs that were digested with different restriction endonucleases that cut the monomer only once at different locations. DNA synthesis begins at a specific site in pBR322 and progresses in a uni-directional manner. Thus, the main difference between these sets of RIs was the relative position of the origin. The results obtained clearly showed that populations of RIs containing an internal bubble where initiation occurred at different relative positions do not generate signals that co-migrate all-the-way in 2D gels. Despite this observation, however, our results support the notion that random initiation is indeed responsible for the peculiar 'bubble' signal observed in the case of several metazoan eukaryotes.

Conserved features in the mode of replication of eukaryotic ribosomal RNA genes.

It was previously shown that a 1.5 kb fragment located in the non-transcribed spacer (NTS) is the earliest replicating region of pea (Pisum sativum) rDNA in synchronized root cells. In the present report the structure of this region was characterized. It contains a cluster of four 11 bp near matches to the Saccharomyces cerevisiae ARS consensus sequence (ACS). These near matches are embedded in an A+T rich domain located upstream from the transcription initiation site. We identified and mapped an intrinsic DNA bending locus 5' to the cluster of near matches. Several eukaryotic origins including the ARS from the budding yeast show very similar structural features. This observation strengthens the notion that pea rDNA replication initiates at or near this region. Replication of the entire pea rDNA repeat was analysed by two-dimensional (2D) agarose gel electrophoresis. The results obtained indicate that only a small fraction of the potential origins is used in each replication round. Forks moving in the direction opposite to rRNA transcription are stalled at a polar replication fork barrier (RFB), which mapped near the 3' end of the transcription unit. Consequently, most of pea rDNA appears to replicate in a unidirectional manner. These results show that the strategy used to replicate pea and yeast rRNA genes is very similar, suggesting that it has been conserved and might be common to most eukaryotes.

Initiation of DNA replication in ColE1 plasmids containing multiple potential origins of replication.

We have investigated the frequency of replication origin usage in bacterial plasmids containing more than one potential origin. Escherichia coli recA- cells were selectively transformed with pBR322 monomers, dimers, or trimers. Plasmid DNA was isolated and digested with a restriction enzyme that cut the monomer only once, and the replicative intermediates (RIs) were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis. Evidence for initiation outside the linearized plasmid was found only for oligomers. Moreover, in dimers, the intensity of the signal indicative for external initiation was equivalent to that reflecting internal initiation, whereas it was approximately twice as strong in trimers. To determine whether initiation could occur simultaneously at two origins in a single plasmid, we studied the replication of a neodimer in which both units could be unambiguously distinguished. The results showed that although both origins were equally competent to initiate replication, only one was active per plasmid. These observations strongly suggest that in ColE1 plasmids, replication initiates at a single site even when there are several identical potential origins per plasmid. In addition to the conventional two-dimensional gel patterns, novel specific patterns were observed with intensities that varied from one DNA sample to another. These unique patterns were the result of breakage of the RIs at a replication fork. This type of breakage changes both the mass and shape of RIs. When the entire population of RIs is affected, a new population of molecules is formed that may generate a novel pattern in two-dimensional gels.

Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis.

Two-dimensional (2D) agarose gel electrophoresis is progressively replacing electron microscopy as the technique of choice to map the initiation and termination sites for DNA replication. Two different versions were originally developed to analyze the replication of the yeast 2 microns plasmid. Neutral/Neutral (N/N) 2D agarose gel electrophoresis has subsequently been used to study the replication of other eukaryotic plasmids, viruses and chromosomal DNAs. In some cases, however, the results do not conform to the expected 2D gel patterns. In order to better understand this technique, we employed it to study the replication of the colE1-like plasmid, pBR322. This was the first time replicative intermediates from a unidirectionally replicated plasmid have been analyzed by means of N/N 2D agarose gel electrophoresis. The patterns obtained were significantly different from those obtained in the case of bidirectional replication. We showed that identification of a complete are corresponding to molecules containing an internal bubble is not sufficient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin. We also showed that unidirectionally replicated fragments containing a stalled fork can produce a pattern with an inflection point. Finally, replication appeared to initiate at only some of the potential origins in each multimer of pBR322 DNA.