Generation of supercoils in nicked and gapped DNA drives DNA unknotting and postreplicative decatenation

Abstract

Due to the helical structure of DNA the process of DNA replication is topologically complex. Freshly replicated DNA molecules are catenated with each other and are frequently knotted. For proper functioning of DNA it is necessary to remove all of these entanglements. This is done by DNA topoisomerases that pass DNA segments through each other. However, it has been a riddle how DNA topoisomerases select the sites of their action. In highly crowded DNA in living cells random passages between contacting segments would only increase the extent of entanglement. Using molecular dynamics simulations we observed that in actively supercoiled DNA molecules the entanglements resulting from DNA knotting or catenation spontaneously approach sites of nicks and gaps in the DNA. Type I topoisomerases, that preferentially act at sites of nick and gaps, are thus naturally provided with DNA-DNA juxtapositions where a passage results in an error-free DNA unknotting or DNA decatenation.

Figure 1.

Arising supercoiling pushes trefoil knot out of the linear DNA. Snapshots from one continuous molecular dynamics simulation (see methods) where gyrase starts introducing negative supercoiling into knotted linear DNA that is ca 3 kb long. The site of active swivel (gyrase) is shown as the blue bead. (A) Starting configuration of knotted linear DNA molecule. (B) Formation of plectonemes starts in the vicinity of active gyrase. (C, D). As plectonemically wound region increases it effectively pushes DNA segments that intervene with formation of relatively regular interwound regions. This process induces slithering of the knotted portion of the molecule towards the ends of knotted linear DNA and leads to its unknotting.

Figure 2.

Arising supercoiling in nicked circular DNA pushes the trefoil knot towards the site of the nick. (A). A thermalized configuration of trefoil-forming nicked DNA molecule with ca 3000 bp with the sites of active swivel (gyrase) and passive swivel (nick) indicated as blue and reddish beads, respectively. (B, C) Working gyrase progressively confines the knotted portion and pushes it towards the site of the nick. (D) A snapshot from the steady state situation where the knot is localized in the vicinity of the nick and where the rate of supercoil generation by the gyrase is equal to the rate of supercoil dissipation at the site of the nick. Notice that knotted portion is highly localized and highly curved.

Figure 3.

Pushing of DNA knots towards nicks and gaps does not require antipodal locations of active and passive swivel sites and it applies to all tested knot types. (A, B) snapshots of the simulation where active and passive swivel sites (indicated as blue and reddish beads, respectively) are placed 90° apart on the circular map of 3 kb large plasmid forming a trefoil knot. (C, D) snapshots from the simulation where a complex knot with 8 crossings (8₁₈) is pushed towards the nick site. Notice that also in this case the knot is progressively tightened and localizes in the vicinity of a nick site (reddish bead). Inset shows 8₁₈ knot in a form with its minimal number of perceived crossings.

Figure 4.

Juxtapositions between gaps and duplex regions in confined knots are perfectly suited for topoIII unknotting. Snapshots from the steady state situation where continuous rotations at the active swivel site (blue bead) generate supercoils confining and pushing the trefoil knot towards the site of a short gap (indicated as a green region). Notice on a close up views that gapped region bends over a duplex region located within the tightened knot. This specific structure results from the fact that single stranded DNA regions are very flexible and show decreased electrostatic repulsion since the number of charges in ssDNA is twice smaller than in dsDNA.

Figure 5.

Model of DNA unknotting and decatenation involving long distance cooperation between DNA gyrase and topoIII. Simulation snapshots illustrating how gyrase (blue bead) that introduces DNA supercoiling and topoisomerase III acting at the site of gaps (green region) can act together in the process of DNA unknotting (A) and DNA decatenation (B). To simulate the process of topoIII mediated passages occurring at short gaps we have removed self-avoidance between gaps and the rest of modelled DNA molecules. Blue arcs in (A) and (B) indicate the directions of movements of double stranded regions that were allowed to pass through single stranded gaps as that would be the case of topoIII-mediated passages.