Abstract
Two-dimensional agarose gel electrophoresis is the method of choice to identify and quantify all the topological forms DNA molecules can adopt in vivo. Here we describe the materials and protocols needed to analyze catenanes, the natural outcome of DNA replication, in Saccharomyces cerevisiae. We describe the formation of pre-catenanes during replication and how inhibition of topoisomerase 2 leads to the accumulation of intertwined sister duplexes. This knowledge is essential to determine how replication forks blockage or pausing affects the dynamic of DNA topology during replication.
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References
Schvartzman JB, Stasiak A (2004) A topological view of the replicon. EMBO Rep 5:256–261
Champoux JJ, Been MD (1980) Topoisomerases and the swivel problem. In: Alberts B (ed) Mechanistic studies of DNA replication and genetic recombination, ICN-UCLA symposia on molecular and cellular biology. Academic Press, New York, pp 809–815
Cebrián J, Castán A, Martínez V, Kadomatsu-Hermosa MJ, Parra C, Fernández-Nestosa MJ, Schaerer C, Hernández P, Krimer DB, Schvartzman JB (2015) Direct evidence for the formation of precatenanes during DNA replication. J Biol Chem 290:13725–13735
Schalbetter SA, Mansoubi S, Chambers AL, Downs JA, Baxter J (2015) Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability. Proc Natl Acad Sci U S A 112:4565–4570
Adams DE, Shekhtman EM, Zechiedrich EL, Schmid MB, Cozzarelli NR (1992) The role of topoisomerase-IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication. Cell 71:277–288
Baxter J, Diffley JF (2008) Topoisomerase II inactivation prevents the completion of DNA replication in budding yeast. Mol Cell 30:790–802
Baxter J, Sen N, Martínez VL, De Carandini ME, Schvartzman JB, Diffley JF, Aragón L (2011) Positive supercoiling of mitotic DNA drives decatenation by topoisomerase II in eukaryotes. Science 331:1328–1332
Martínez-Robles ML, Witz G, Hernández P, Schvartzman JB, Stasiak A, Krimer DB (2009) Interplay of DNA supercoiling and catenation during the segregation of sister duplexes. Nucleic Acids Res 37:5126–5137
Sundin O, Varshavsky A (1980) Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers. Cell 21:103–114
Sundin O, Varshavsky A (1981) Arrest of segregation leads to accumulation of highly intertwined catenated dimers dissection of the final stages of SV40 DNA replication. Cell 25:659–669
Cebrián J, Monturus E, Martínez-Robles ML, Hernández P, Krimer DB, Schvartzman JB (2014) Topoisomerase 2 is dispensable for the replication and segregation of small yeast artificial chromosomes (YACs). PLoS One 9:e104995
Murray AW, Szostak JW (1983) Construction of artificial chromosomes in yeast. Nature 305:189–193
Martín-Parras L, Lucas I, Martínez-Robles ML, Hernández P, Krimer DB, Hyrien O, Schvartzman JB (1998) Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis. Nucleic Acids Res 26:3424–3432
Acknowledgments
This work was supported by grant BFU2014-56835 from the Spanish Ministerio de Economía y Competitividad to JBS. We thank Jonathan Baxter and Luis Aragón for plasmids and DNA sequence data. The authors also acknowledge Alicia Rodríguez-Bernabé for technical help.
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Castán, A., Hernández, P., Krimer, D.B., Schvartzman, J.B. (2018). DNA Catenation Reveals the Dynamics of DNA Topology During Replication. In: Drolet, M. (eds) DNA Topoisomerases. Methods in Molecular Biology, vol 1703. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7459-7_5
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DOI: https://doi.org/10.1007/978-1-4939-7459-7_5
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