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

ABSTRACT: 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.

SUBMITTER: Cebrian J

PROVIDER: S-EPMC8615216 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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

Cebrián Jorge J Martínez Victor V Hernández Pablo P Krimer Dora B DB Fernández-Nestosa María-José MJ Schvartzman Jorge B JB

Biology 20211117 11

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: t ...[more]

PMID: 34827187

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Project description:BackgroundProteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF) that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE) is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly.ResultsThe proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit revealed clearer presentation of the isoforms and increased intensities of the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gel images as compared with untreated SF samples and SF samples treated with acetone.ConclusionsThe acetone precipitation method and the combined treatment effect of acetone and 2-DE Clean-Up Kit are not preferred in preparing SF samples for 2-DE analysis as both protein intensities and numbers decrease significantly. On the other hand, 2-D Clean-Up Kit treated SF samples revealed clearer isoforms and higher intensities for the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gels. As a result, it is recommended that SF samples should be treated with protein clean up products such as 2-D Clean-Up Kit first before conducting proteomic research in searching for the relevant biomarkers associated with knee osteoarthritis.

Dataset Information

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

Publications

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

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