Direct Evidence for the Formation of Precatenanes During DNA Replication [Cell Biology]

March 31st, 2015 by Cebrian, J., Castan, A., Martinez, V., Kadomatsu–Hermosa, M.–J., Parra, C., Fernandez–Nestosa, M. J., Schaerer, C., Hernandez, P., Krimer, D. B., Schvartzman, J. B.

The dynamics of DNA topology during replication is 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 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 DNA underwound. Putative intertwines of sister duplexes in the replicated region are called precatenanes. Fork swiveling and the formation of precatenanes, though, 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.