Supercoiling, knotting and replication fork reversal in partially replicated plasmids
| Autor: | Pablo E. Hernández, Leticia Olavarrieta, Jorge Bernardo Schvartzman, José Sogo, Andrzej Stasiak, Dora B. Krimer, María Luisa Martínez-Robles |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
DNA Replication
Replication fork reversal Catenated dimers Topoisomer Escherichia-coli Biology Article Intertwined chemistry.chemical_compound Plasmid Ethidium Escherichia coli Genetics Agarose-gel electrophoresis Electrophoresis Gel Two-Dimensional Models Genetic DNA Superhelical DNA replication DNA Replication Fork Molecular biology Recombination SV40 DNA-replication Blotting Southern Microscopy Electron chemistry Potential origins DNA Superhelical/biosynthesis DNA Superhelical/chemistry Escherichia coli/genetics Nucleic Acid Conformation Plasmids/biosynthesis Plasmids/chemistry Biophysics DNA supercoil Ethidium bromide DNA Termination Plasmids |
| Zdroj: | Nucleic Acids Research, vol. 30, no. 3, pp. 656-666 Scopus-Elsevier Digital.CSIC. Repositorio Institucional del CSIC instname |
| Popis: | 11 páginas, 8 figuras, -- PAGS nros.656-666 To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive DeltaLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide. PM 9701-38 PGC PB98048 from the Spanish CICYT, 99/0850 from the Spanish FIS, 08.6/0016/2007 from CAM, and from the Swiss National Science Foundation to JMS (31-63418), and A.S. (31-61636)and (31-58841). |
| Databáze: | OpenAIRE |
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