Summary
A fundamental process in DNA replication is the disentangling of the two parental strands by DNA topoisomerases. In this chapter, I detail the topological analysis of plasmid replication intermediates using two-dimensional (2D) agarose gels. The method can resolve replication intermediates according to mass and topology, and can resolve unlinked monomeric circles from catenated dimers of varying topology. The method has been used, alone or in combination with a procedure for purifying covalent protein–DNA complexes, to analyse the effect of topoisomerase inhibitors on the topology of replication intermediates, to map the location of drug-stabilized topoisomerase cleavage complexes with respect to replication forks and to detect the breakage and repair of replication forks following collision with cleavage complexes. Other applications include the detection of knots that form independently of, or concomitantly with, DNA replication.
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Acknowledgments
The author thanks Torsten Krude, Arach Goldar, and Cyrille le Breton for critical reading of the manuscript and I. Lucas and T. Germe for the autoradiograms shown in Figs. 8 and 9, respectively. Work in my laboratory is supported by the Association pour la Recherche sur le Cancer, the Ligue Nationale contre le Cancer (Comité de Paris), the Agence Nationale pour la Recherche, and the Fondation pour la Recherche Médicale.
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Hyrien, O. (2009). Topological Analysis of Plasmid DNA Replication Intermediates Using Two-Dimensional Agarose Gels. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 521. Humana Press. https://doi.org/10.1007/978-1-60327-815-7_8
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DOI: https://doi.org/10.1007/978-1-60327-815-7_8
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