Supercoiling, knotting and replication fork reversal in partially replicated plasmids.

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Serval ID
serval:BIB_6F7F681F761D
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Supercoiling, knotting and replication fork reversal in partially replicated plasmids.
Journal
Nucleic Acids Research
Author(s)
Olavarrieta L., Martínez-Robles M.L., Sogo J.M., Stasiak A., Hernández P., Krimer D.B., Schvartzman J.B.
ISSN
1362-4962[electronic], 0305-1048[linking]
Publication state
Published
Issued date
02/2002
Volume
30
Number
3
Pages
656-666
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Abstract
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.
Keywords
Blotting, Southern, DNA Replication, DNA, Superhelical/biosynthesis, DNA, Superhelical/chemistry, Electrophoresis, Gel, Two-Dimensional, Escherichia coli/genetics, Ethidium, Microscopy, Electron, Models, Genetic, Nucleic Acid Conformation, Plasmids/biosynthesis, Plasmids/chemistry
Pubmed
Web of science
Open Access
Yes
Create date
24/01/2008 11:36
Last modification date
25/09/2019 7:09
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