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oai:docta.ucm.es:20.500.14352/47442023-08-25T10:48:44Zcom_20.500.14352_14col_20.500.14352_15
Docta Complutense author Cebrián Castillo, Jorge author Martínez, Victor author Hernández, Pablo author Krimer, Dora B. author Fernández Nestosa, María José author Schvartzman, Jorge B. 2023-06-16T14:20:28Z 2023-06-16T14:20:28Z 2021-11-13 2079-7737 10.3390/biology10111195 https://hdl.handle.net/20.500.14352/4744 https://doi.org/10.3390/biology10111195 https://www.mdpi.com/2079-7737/10/11/1195/htm DNA topoisomerases are the enzymes that regulate DNA topology in all living cells. Since the discovery and purification of ω (omega), when the first were topoisomerase identified, the function of many topoisomerases has been examined. However, their ability to relax supercoiling and unlink the pre-catenanes of partially replicated molecules has received little attention. Here, we used two-dimensional agarose gel electrophoresis to test the function of three type II DNA topoisomerases in vitro: the prokaryotic DNA gyrase, topoisomerase IV and the human topoisomerase 2α. We examined the proficiency of these topoisomerases on a partially replicated bacterial plasmid: pBR-TerE@AatII, with an unidirectional replicating fork, stalled when approximately half of the plasmid had been replicated in vivo. DNA was isolated from two strains of Escherichia coli: DH5αF’ and parE10. These experiments allowed us to assess, for the first time, the efficiency of the topoisomerases examined to resolve supercoiling and pre-catenanes in partially replicated molecules and fully replicated catenanes formed in vivo. The results obtained revealed the preferential functions and also some redundancy in the abilities of these DNA topoisomerases in vitro. eng Atribución 3.0 España Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates journal article URL https://docta.ucm.es/bitstreams/000a7d31-aaef-4990-8c3d-7a98a35a71ce/download File MD5 104652e6c106da8d092ef7d41d5b8fd1 1834970 application/pdf biology-10-01195.pdf