A topological view of the replicon

Abstract

The replication of circular DNA faces topological obstacles that need to be overcome to allow the complete duplication and separation of newly replicated molecules. Small bacterial plasmids provide a perfect model system to study the interplay between DNA helicases, polymerases, topoisomerases and the overall architecture of partially replicated molecules. Recent studies have shown that partially replicated circular molecules have an amazing ability to form various types of structures (supercoils, precatenanes, knots and catenanes) that help to accommodate the dynamic interplay between duplex unwinding at the replication fork and DNA unlinking by topoisomerases.

Figure 1

Supercoiling: its handedness and sign. (A) Negatively supercoiled DNA (left) loses supercoiling due to local DNA unwinding mediated by DNA helicases (shown as grey wedges) and then becomes (+) supercoiled by further strand separation. Notice that the intertwined superhelix is right-handed in (−) supercoiled molecules and left-handed in (+) supercoiled ones. The sign of the duplex–duplex crossings (see panel B) changes from (−) to (+) upon a change from negative to positive supercoiling. (B) Topological convention of sign assignment of perceived crossings. In a (−) crossing, one would need to turn the overlying direction arrow clockwise to align it with the underlying direction arrow (the rotation needs to be smaller than 180°). In a (+) crossing the required rotation would be counterclockwise. Notice that orientation of the underlying and overlying direction arrows at each crossing are not independent from each other but result from assigning a consistent direction along the whole DNA molecule analysed. To facilitate sign recognition in A and B, the overlying and underlying direction arrows are marked in red and blue, respectively.

Figure 2

Topological sign and handedness of duplex–duplex intertwining in supercoiled replication intermediates. (A) Schematic drawing of (−) and (+) supercoiled replication intermediates (RIs). (B) Elastic transition between toroidally wound (around core histones for example) and intertwined form of (−) supercoiled DNA. Notice that, in the toroidally wound form, the segments that cross in a projection run in the same direction around a virtual torus, whereas, in the intertwined form, the crossing segments run in opposite directions around the virtual cylinder enclosed by the DNA. This change of relative orientation causes the topological signs to remain the same despite a perceived change from left- to right-handed winding of the superhelices. The mathematical convention applied in DNA topology assigns a parallel orientation to both strands of DNA (this is required to have a (+) linking number in B-DNA, which forms a right-handed helix; Bates & Maxwell, 1993). For this reason, to trace the linking number contribution of parental strands in an RI, one needs to assign the same direction to both newly replicated duplex regions. In (−) supercoiled DNA there is a tendency to release the torsional stress by left-handed winding of unpaired strands or by flipping runs of alternating purine–pyrimidine from right-handed B-DNA to the left-handed Z-DNA (DiCapua et al, 1983). It is therefore energetically favourable in deproteinized (−) supercoiled RIs that the newly synthesized duplex regions are wound around each other in a left-handed way. The opposite situation applies to (+) supercoiled RIs. The parental duplex is indicated in blue and green, whereas nascent strands are depicted in red.

Figure 3

The topological cycle of a replicon. (A) Unreplicated (−) supercoiled plasmid. (B) Twenty-five per cent replicated (−) supercoiled RI where the parental duplex winds right-handed, whereas the sister duplexes wind in a left-handed manner. (C) Fifty per cent replicated RI where supercoiling is zero. (D) Seventy-five per cent replicated (+) supercoiled RI where the parental duplex winds left-handed, whereas the sister duplexes wind in a right-handed manner. (E) Seventy-five per cent replicated (−) supercoiled RI where the parental duplex winds right-handed, whereas the sister duplexes wind in a left-handed manner. (F) One hundred per cent replicated catenane. (E') Seventy-five per cent replicated (−) supercoiled RI bearing a knotted replication bubble. (D') Seventy-five per cent replicated RI where supercoiling is zero containing two branched four-way Holliday-like junctions, called 'chicken-foot' structures. Red arrows indicate the putative most frequent pathway. Grey arrows show alternative pathways. The parental duplex is indicated in blue and green, whereas nascent strands are depicted in red.