Abstract
The dynamics of DNA topology during replication are still poorly understood. Bacterial plasmids are negatively supercoiled. This underwinding facilitates strand separation of the DNA duplex during replication. Leading the replisome, a DNA helicase separates the parental strands that are to be used as templates. This strand separation causes overwinding of the duplex ahead. If this overwinding persists, it would eventually impede fork progression. In bacteria, DNA gyrase and topoisomerase IV act ahead of the fork to keep DNA underwound. However, the processivity of the DNA helicase might overcome DNA gyrase and topoisomerase IV. It was proposed that the overwinding that builds up ahead of the fork could force it to swivel and diffuse this positive supercoiling behind the fork where topoisomerase IV would also act to maintain replicating the DNA underwound. Putative intertwining of sister duplexes in the replicated region are called precatenanes. Fork swiveling and the formation of precatenanes, however, are still questioned. Here, we used classical genetics and high resolution two-dimensional agarose gel electrophoresis to examine the torsional tension of replication intermediates of three bacterial plasmids with the fork stalled at different sites before termination. The results obtained indicated that precatenanes do form as replication progresses before termination.
Highlights
Changes in DNA topology during replication are still poorly understood
In the case of unimpaired replication, fully replicated molecules would end up catenated in an RH manner solely due to the linking of the last ϳ200 bp of DNA for which it would not be possible for DNA gyrase and topoisomerase IV (Topo IV) to remove the links ahead of the replication fork at termination [6, 17]
To check whether the topology of bacterial plasmids was somehow affected in DH5␣FЈ cells in the absence of norfloxacine compared with a wild-type strain, we determined the supercoiling density () of unreplicated forms of pBRterE@AatII isolated from either DH5␣FЈ cells that are RecAϪ, Topo IVϩ, and gyrA96 or W3110 cells that are RecAϩ, Topo IVϩ, and Gyrϩ
Summary
Changes in DNA topology during replication are still poorly understood. Results: Classical genetics and two-dimensional agarose gel electrophoresis showed that RIs tensioned in the absence of Topo IV. In the case of unimpaired replication, fully replicated molecules would end up catenated in an RH manner solely due to the linking of the last ϳ200 bp of DNA for which it would not be possible for DNA gyrase and Topo IV to remove the links ahead of the replication fork at termination [6, 17] According to this hypothesis, if RIs with the fork stalled at the ter-Tus complex were isolated from bacterial cells where Topo IV is active, molecules with the fork stalled would be (Ϫ)-supercoiled (showing RH crossings) in the unreplicated region and devoid of precatenanes (Fig. 2A). DNA gyrase would keep introducing (Ϫ)-supercoils in the unreplicated region, and to keep the torsional tension equilibrated, these RH crossings from the unreplicated region would diffuse to the replicated one as LH precatenanes, where Topo IV would progressively remove them. It would mainly include (Ϫ)-supercoiling for pBR-terE@StyI and predominantly left-handed precatenanes for pBR-terE@DraI (see Fig. 3)
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