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

L Olavarrieta1, M L Martínez-Robles, J M Sogo

  • 1Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas (CSIC), Velázquez 144, 28006 Madrid, Spain.

Nucleic Acids Research
|January 26, 2002
PubMed
Summary

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Replication forks halt at the Ter-TUS complex, creating unique DNA structures. These partially replicated plasmids absorb supercoiling differently, sometimes through replication fork reversal.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The Escherichia coli Ter-TUS system is crucial for regulating DNA replication termination.
  • Understanding the topological changes in partially replicated plasmids is essential for comprehending DNA replication dynamics.

Purpose of the Study:

  • To investigate the structural and topological characteristics of partially replicated plasmids containing the TerE terminator.
  • To elucidate the mechanism by which partially replicated plasmids interact with supercoiling and replication fork dynamics.

Main Methods:

  • Cloning of the TerE terminator into the pBR18 ColE1 vector at specific sites.
  • Analysis using neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy.
  • Assessment of supercoiling properties in the presence of intercalating agents like chloroquine and ethidium bromide.

Related Experiment Videos

Main Results:

  • Accumulation of specific replication intermediates with increased mass (1.26x and 1.57x) due to replication fork arrest at the Ter-TUS complex.
  • Changes in plasmid topology, including knotted bubbles and altered electrophoretic mobility, were observed in partially replicated molecules.
  • Partially replicated plasmids showed reduced ability to sustain positive supercoiling, with evidence of replication fork reversal, particularly in the presence of chloroquine.

Conclusions:

  • The Ter-TUS complex effectively halts replication forks, leading to distinct topological states in partially replicated plasmids.
  • Partially replicated plasmids exhibit altered supercoiling behavior, potentially involving replication fork regression as a compensatory mechanism.
  • The findings provide insights into the complex interplay between DNA replication, termination, and topological stress in plasmids.