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DNA dynamics during replication: the benefit of entanglement Schvartzman, Jorge


During replication, DNA molecules undergo topological changes that affect supercoiling, catenation and knotting. To better understand this process and the role of topoisomerases, the enzymes that control DNA topology in vivo, high resolution two- dimensional agarose gel electrophoresis (2Dgels) and atomic force microscopy (AFM) were used to examine partially replicated bacterial plasmids containing replication forks stalled at specific sites. The exposure of these replication intermediates (RIs) isolated from Escherichia coli mutant cells proficient or deficient for Topo IV to various topoisomerases in vitro generated surprising observations. The results obtained together with computer simulations based on Metropolis Monte Carlo helped us to predict the thermodynamic stability of the molecules and to determine the potential energy that can be stored in the replicated and unreplicated regions. Altogether, these observations strongly suggest that type II DNA topoisomerases recognize the geometry of DNA duplex crossings, probably throughout their interaction with other proteins. The geometry of the crosses differs between the unreplicated and replicated regions, changes continuously as replication forks advance and is dramatically altered by deproteinization. Work in collaboration with Jorge Cebrián,Víctor Martínez, María José Fernández, Christian Schaerer, Pablo Hernández, Dora B. Krimer, Jorge B. Schvartzman.

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