Cartoon representing different forms of catenanes after projection on a plane. (A) One pair of nicked DNA rings catenated once. (B) One pair of nicked DNA rings catenated twice. (C) One pair of DNA rings catenated once where one ring is nicked and the other covalently closed and supercoiled. (D) One pair of DNA rings catenated once where both rings are covalently closed and supercoiled. n = nick; OC = open circle; CCC = covalently closed circle; Ca = catenation number. Parental DNA strands are depicted in blue and green while newly synthesized strands are depicted in red.

Preferential inhibition of Topo IV leads to the accumulation of DNA catenanes. Autoradiograms of 2D gels corresponding to pBR18 isolated from DH5αF′ E. coli cells untreated (A) and after exposure to norfloxacin (B). A diagrammatic interpretation of the different signals in the autoradiograms is shown to the right (C) where CatAs are depicted in blue, CatBs in red and CatCs in green. RIs = nicked replication intermediates; OCd = open circles corresponding to dimers; OCm = open circles corresponding to monomers; Knm = nicked knotted monomers; Ld = linearized dimers; Lm = linearized monomers. Monomeric topoisomers are depicted in black.

Partial and complete nicking allows identification of all forms of catenanes. Autoradiograms of 2D gels corresponding to pBR18 isolated from DH5αF′ E. coli cells after exposure to norfloxacin and complete (A) or partial (B) digestion with the single-stranded DNA nicking enzyme Nb-BsmI. A diagrammatic interpretation of the autoradiograms is shown to their right where CatAs are depicted in blue, CatBs in red and CatCs in green. A red arrow in (B) points to a distinct inflection that was evidenced for CatBs when Ca reached 10–12. RIs = nicked replication intermediates; Knd = nicked knotted dimers; OCd = open circles corresponding to dimers; OCm = open circles corresponding to monomers; Knm = nicked knotted monomers; Ld = linearized dimers; Lm = linearized monomers. Monomeric topoisomers are depicted in black.

The free energy of DNA supercoiling increases with the extent of DNA catenation. (A) Profiles of the free energy of DNA supercoiling i.e. the energy that would be liberated upon nicking of the catenated DNA with various levels of catenation (Ca) and various extent of supercoiling (ΔLk) for modelled DNA molecules of ∼4400 bp. Notice that the free energy of catenation is not included in the graph since we are not considering here the energy that would be liberated upon progressive decatenation of modelled catenanes by Topo IV. (B) Free energy costs that DNA gyrase would need to overcome to perform sequential supercoiling cycles in catenated DNA molecules with ∼4400 bp that have different catenation numbers. Notice that during each catalytic cycle DNA gyrase reduces the linking number by two, notice also that each sequential cycle requires more energy and that in more complex catenanes this increase is more rapid than in less complex ones. The dashed line indicates the estimated level to which DNA gyrase would be able to supercoil protein-free DNA rings with different extents of catenation.

Limited levels of (−) supercoiling decompacts highly interlinked catenanes. Smoothed snapshots of equilibrated configurations of catenanes with Ca = 19, which component rings are either free of torsional tension (ΔLk = 0) (A) or are mildly (−) supercoiled (ΔLk = −5) (B). (A) Highly interlinked catenane with component rings that are free of torsional tension adopt a twisted configuration since this lowers their elastic energy. The same conclusion was reached in simulation studies of highly linked nicked DNA circles (20). (B) Introduction of torsional stress, which would have resulted from two catalytic cycles of DNA gyrase on each of torsionally relaxed but covalently closed component DNA rings, brings the catenanes into a more open configuration with a more regular toroidal structure.

In CatBs and CatCs interlinking and supercoiling compete with each other. Autoradiograms of 2D gels corresponding to pBR18 isolated from DH5αF′ E. coli cells after exposure to norfloxacin to accumulate catenated forms. The second dimension occurred in the presence of different concentrations of chloroquine and the autoradiograms were aligned so that OCs and Ls, whose electrophoretic mobility is not significantly affected by chloroquine, coincided. A diagrammatic interpretation of each autoradiogram is shown below where CatAs are depicted in blue, CatBs in red and CatCs in green. In the diagrams corresponding to 10 and 40 µg/ml chloroquine, CatCs where Ca = 3–12 were drawn as a continuous line as individual spots were difficult to identify at the resolution of these photographs. Monomeric topoisomers are depicted in black. OCRIs = nicked replication intermediates; OCd = open circles corresponding to dimers; OCm = open circles corresponding to monomers; Ld = linearized dimers; Lm = linearized monomers. Red arrows point to CatBs with the lowest Ca value (10–12) that chloroquine was unable to decompose in topoisomers during the second dimension.

Structure of DNA catenanes with decreasing catenation numbers and expected levels of DNA supercoiling that DNA gyrase could introduce. Smoothed snapshots from Metropolis–Monte Carlo simulations of DNA catenanes representing progressive stages of decatenation process under conditions where the action of DNA gyrase maintains the DNA at supercoiling level set by a quasi-constant energy input that DNA gyrase can use for every catalytic cycle under in vivo conditions. (A) Stage with Ca = 19. Winding of the two molecules around each other is distributed along the entire length while the intramolecular plectonemes due to supercoiling do not form yet. (B) Stage with Ca = 13. Regions with intramolecular plectonemic supercoiling start to be visible and they correspond to regions where the two rings do not wind around each other. (C) Stage with Ca = 7. The region where the two rings wind around each other becomes confined while the regions with plectonemic supercoiling appear like typical branches in supercoiled DNA (25). (D) Stage with Ca = 1. Component rings have the typical appearance of plectonemically supercoiled DNA molecules while catenation preferentially involves apical loops of supercoiled DNA (48).

Cartoon illustrating the transition from a highly intertwined non-supercoiled catenane (A) to a singly catenated and highly supercoiled one (D). Just after completion of DNA replication both sister duplexes are highly intertwined and share a common axis. These type C torus catenanes accept a low level of DNA supercoiling that does not yet manifest itself in form of extruded plectonemes (A). As decatenation progresses the mechanical stress in DNA molecules decreases and this allows DNA gyrase to introduce more (−) supercoiling that start to manifest itself as extruded plectonemes (B). As the degree of catenation decreases, supercoiling builds-up and type C torus catenanes tend to have winding and supercoiling clearly segregated (C). Finally, intertwining becomes frequently confined to apical portions (48) and the sister duplexes appear highly supercoiled just before their segregation (D). Asterisks denote intermolecular crossings and dots intramolecular crossings. Parental DNA strands are depicted in blue and green while newly synthesized strands are depicted in red.