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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MDPI AG
2021-11-01
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T05:41:49Z |
| format | Article |
| id | doaj.art-f86df036de0b4fda942c8af1ffc489bc |
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| issn | 2079-7737 |
| language | English |
| last_indexed | 2024-03-10T05:41:49Z |
| publishDate | 2021-11-01 |
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| series | Biology |
| spelling | doaj.art-f86df036de0b4fda942c8af1ffc489bc2023-11-22T22:28:44ZengMDPI AGBiology2079-77372021-11-011011119510.3390/biology10111195Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication IntermediatesJorge Cebrián0Victor Martínez1Pablo Hernández2Dora B. Krimer3María-José Fernández-Nestosa4Jorge B. Schvartzman5Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas (CSIC), 28040 Madrid, SpainBioinformatics Laboratory, Polytechnic School, National University of Asunción, San Lorenzo P.O. Box 2111, ParaguayDepartment of Cellular and Molecular Biology, Centro de Investigaciones Biológicas (CSIC), 28040 Madrid, SpainDepartment of Cellular and Molecular Biology, Centro de Investigaciones Biológicas (CSIC), 28040 Madrid, SpainBioinformatics Laboratory, Polytechnic School, National University of Asunción, San Lorenzo P.O. Box 2111, ParaguayDepartment of Cellular and Molecular Biology, Centro de Investigaciones Biológicas (CSIC), 28040 Madrid, SpainDNA 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.https://www.mdpi.com/2079-7737/10/11/1195DNA topologyreplicationsupercoilingpre-catenationtwo-dimensional agarose gel electrophoresis |
| spellingShingle | 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 Biology DNA topology replication supercoiling pre-catenation two-dimensional agarose gel electrophoresis |
| title | 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_short | 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 |
| topic | DNA topology replication supercoiling pre-catenation two-dimensional agarose gel electrophoresis |
| url | https://www.mdpi.com/2079-7737/10/11/1195 |
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