María Luisa Martínez-Robles

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.

Interplay of DNA supercoiling and catenation during the segregation of sister duplexes

The discrete regulation of supercoiling, catenation and knotting by DNA topoisomerases is well documented both in vivo and in vitro, but the interplay between them is still poorly understood. Here we studied DNA catenanes of bacterial plasmids arising as a result of DNA replication in Escherichia coli cells whose topoisomerase IV activity was inhibited. We combined high-resolution two-dimensional agarose gel electrophoresis with numerical simulations in order to better understand the relationship between the negative supercoiling of DNA generated by DNA gyrase and the DNA interlinking resulting from replication of circular DNA molecules. We showed that in those replication intermediates formed in vivo, catenation and negative supercoiling compete with each other. In interlinked molecules with high catenation numbers negative supercoiling is greatly limited. However, when interlinking decreases, as required for the segregation of newly replicated sister duplexes, their negative supercoiling increases. This observation indicates that negative supercoiling plays an active role during progressive decatenation of newly replicated DNA molecules in vivo.

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.

Topo IV is the topoisomerase that knots and unknots sister duplexes during DNA replication

DNA topology plays a crucial role in all living cells. In prokaryotes, negative supercoiling is required to initiate replication and either negative or positive supercoiling assists decatenation. The role of DNA knots, however, remains a mystery. Knots are very harmful for cells if not removed efficiently, but DNA molecules become knotted in vivo. If knots are deleterious, why then does DNA become knotted? Here, we used classical genetics, high-resolution 2D agarose gel electrophoresis and atomic force microscopy to show that topoisomerase IV (Topo IV), one of the two type-II DNA topoisomerases in bacteria, is responsible for the knotting and unknotting of sister duplexes during DNA replication. We propose that when progression of the replication forks is impaired, sister duplexes become loosely intertwined. Under these conditions, Topo IV inadvertently makes the strand passages that lead to the formation of knots and removes them later on to allow their correct segregation.

The benefit of DNA supercoiling during replication.

DNA topology changes dynamically during DNA replication. Supercoiling, precatenation, catenation and knotting interplay throughout the process that is finely regulated by DNA topoisomerases. In the present article, we provide an overview of theoretical and experimental approaches to understand the interplay between various manifestations of topological constraints acting on replicating DNA molecules. Data discussed reveal that DNA entanglements (supercoils and catenanes) play an active role in preventing the formation of deleterious knots.

2D gels and their third-dimension potential

Two-dimensional (2D) agarose gel electrophoresis is one of the most powerful methods to analyze the mass and shape of replication intermediates. It is often use to map replication origins but it is also useful to characterize termination of replication, replication fork barriers and even replication fork reversal. Here, we present protocols, figures and movies with a thorough description of different modes of replication for linear DNA fragments and the corresponding patterns they generate in 2D gels.

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, FOB1Δ, SIR2Δ and the double mutant FOB1ΔSIR2Δ 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 FOB1Δ 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.

Topoisomerase 2 Is Dispensable for the Replication and Segregation of Small Yeast Artificial Chromosomes (YACs)

DNA topoisomerases are thought to play a critical role in transcription, replication and recombination as well as in the condensation and segregation of sister duplexes during cell division. Here, we used high-resolution two-dimensional agarose gel electrophoresis to study the replication intermediates and final products of small circular and linear minichromosomes of Saccharomyces cerevisiae in the presence and absence of DNA topoisomerase 2. The results obtained confirmed that whereas for circular minichromosomes, catenated sister duplexes accumulated in the absence of topoisomerase 2, linear YACs were able to replicate and segregate regardless of this topoisomerase. The patterns of replication intermediates for circular and linear YACs displayed significant differences suggesting that DNA supercoiling might play a key role in the modulation of replication fork progression. Altogether, this data supports the notion that for linear chromosomes the torsional tension generated by transcription and replication dissipates freely throughout the telomeres.