Distortion of DNA junctions imposed by the binding of resolving enzymes: a fluorescence study

J Mol Biol. 2001 Nov 2;313(4):751-64. doi: 10.1006/jmbi.2001.5081.


Junction-resolving enzymes are nucleases that are specific for the structure of the four-way DNA junction. The binding of RuvC of Escherichia coli and Hjc of Sulfolobus solfataricus can be followed by an increase in the fluorescence anisotropy of Cy3 terminally attached to one of the helical arms of a four-way junction. By contrast, there was no change in fluorescein anisotropy with the binding of single dimers of these proteins. Fluorescence resonance energy transfer has therefore been used between fluorescein and Cy3 fluorophores attached to the ends of helical arms to analyse the global structure of the junction on protein binding. The results indicate that both enzymes induce a marked change in the global DNA conformation on the binding of a single dimer. The structure of the protein-junction complexes is independent of the presence or absence of divalent metal ions, unlike that of the protein-free junction. The structures of the RuvC and Hjc complexes are different, but both represent a significant opening of the structure compared to the stacked X-structure of the protein-free junction in the presence of magnesium ions. This protein-induced opening is likely to be important in the function of these enzymes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / metabolism
  • Carbocyanines / metabolism
  • DNA / chemistry*
  • DNA / genetics
  • DNA / metabolism*
  • DNA-Binding Proteins / metabolism
  • Dimerization
  • Electrophoretic Mobility Shift Assay
  • Endodeoxyribonucleases / metabolism*
  • Energy Transfer
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / metabolism
  • Fluorescein / metabolism
  • Fluorescence Polarization*
  • Fluorescent Dyes / metabolism
  • Fluorometry
  • Holliday Junction Resolvases
  • Models, Molecular
  • Nucleic Acid Conformation
  • Protein Binding
  • Recombination, Genetic*
  • Sulfolobus / enzymology*
  • Sulfolobus / genetics


  • Bacterial Proteins
  • Carbocyanines
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • Fluorescent Dyes
  • cyanine dye 3
  • ruvC protein, E coli
  • DNA
  • Endodeoxyribonucleases
  • Holliday Junction Resolvases
  • Fluorescein