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Sequence-dependent bending in plasmid pUC19.

Nancy C Stellwagen1

  • 1Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA. nancy-stellwagen@uiowa.edu

Electrophoresis
|November 5, 2003
PubMed
Summary

The circular permutation assay reveals two stable, sequence-dependent DNA bends in plasmid pUC19. This method reliably detects DNA bending in large DNA molecules.

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Genetics

Background:

  • DNA molecules can adopt bent conformations influenced by their nucleotide sequence.
  • Understanding DNA bending is crucial for processes like transcription and replication.
  • Plasmid DNA, like pUC19, is a common subject for studying DNA structure-function relationships.

Purpose of the Study:

  • To characterize sequence-dependent DNA bending in plasmid pUC19 using the circular permutation assay.
  • To determine the number, location, and stability of DNA bends within the plasmid.
  • To validate the circular permutation assay for analyzing DNA bending in large DNA molecules.

Main Methods:

  • Circular permutation assay on plasmid pUC19 (2686 bp).
  • Electrophoresis in large-pore polyacrylamide gels to analyze mobility patterns of DNA isomers.
  • Investigation of buffer conditions (pH, ions like Mg(++), Ca(++), Ba(++), Zn(++)) and their effect on DNA mobility.
  • Modification of plasmid structure using restriction enzymes and religation.

Main Results:

  • Plasmid pUC19 exhibits two distinct sequence-dependent DNA bends, located at approximately 806 bp and 2617 bp.
  • Mobility patterns are stable across various buffer conditions, indicating inherent DNA helix bends.
  • Divalent cations (Mg(++), Ca(++)) induce conformational changes and alter bend locations, highlighting ionic influence on DNA structure.
  • The number of observed mobility maxima accurately reflects the number of stable DNA bends.

Conclusions:

  • The circular permutation assay is a reliable method for detecting and analyzing sequence-dependent DNA bends in kilobase-sized DNA molecules.
  • Plasmid pUC19 possesses stable, sequence-dictated bends crucial for its structural integrity.
  • Ionic environment significantly influences DNA conformation and the precise location of bends.

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