Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates

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-c...

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Autores principales:	Jorge Cebrián, Victor Martínez, Pablo Hernández, Dora B. Krimer, María-José Fernández-Nestosa, Jorge B. Schvartzman
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	DNA topology replication supercoiling pre-catenation two-dimensional agarose gel electrophoresis Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/f86df036de0b4fda942c8af1ffc489bc
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spelling	oai:doaj.org-article:f86df036de0b4fda942c8af1ffc489bc2021-11-25T16:47:53ZTwo-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates10.3390/biology101111952079-7737https://doaj.org/article/f86df036de0b4fda942c8af1ffc489bc2021-11-01T00:00:00Zhttps://www.mdpi.com/2079-7737/10/11/1195https://doaj.org/toc/2079-7737DNA 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-<i>TerE</i>@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 <i>Escherichia coli</i>: 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.Jorge CebriánVictor MartínezPablo HernándezDora B. KrimerMaría-José Fernández-NestosaJorge B. SchvartzmanMDPI AGarticleDNA topologyreplicationsupercoilingpre-catenationtwo-dimensional agarose gel electrophoresisBiology (General)QH301-705.5ENBiology, Vol 10, Iss 1195, p 1195 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	DNA topology replication supercoiling pre-catenation two-dimensional agarose gel electrophoresis Biology (General) QH301-705.5
spellingShingle	DNA topology replication supercoiling pre-catenation two-dimensional agarose gel electrophoresis Biology (General) QH301-705.5 Jorge Cebrián Victor Martínez Pablo Hernández Dora B. Krimer María-José Fernández-Nestosa Jorge B. Schvartzman Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates
description	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-<i>TerE</i>@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 <i>Escherichia coli</i>: 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.
format	article
author	Jorge Cebrián Victor Martínez Pablo Hernández Dora B. Krimer María-José Fernández-Nestosa Jorge B. Schvartzman
author_facet	Jorge Cebrián Victor Martínez Pablo Hernández Dora B. Krimer María-José Fernández-Nestosa Jorge B. Schvartzman
author_sort	Jorge Cebrián
title	Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates
title_short	Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates
title_full	Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates
title_fullStr	Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates
title_full_unstemmed	Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates
title_sort	two-dimensional gel electrophoresis to study the activity of type iia topoisomerases on plasmid replication intermediates
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/f86df036de0b4fda942c8af1ffc489bc
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Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates

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