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

J M Fogg, M Kvaratskhelia, M F White, D M J Lilley

Research output: Contribution to journalArticlepeer-review

32 Citations (Scopus)

Abstract

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. (C) 2001 Academic Press.

Original languageEnglish
Pages (from-to)751-764
Number of pages14
JournalJournal of Molecular Biology
Volume313
DOIs
Publication statusPublished - 2 Nov 2001

Keywords

  • recombination
  • holliday junction
  • FRET
  • DNA structure
  • DNA-protein interaction
  • RESONANCE ENERGY-TRANSFER
  • CLEAVES HOLLIDAY JUNCTIONS
  • T4 ENDONUCLEASE-VII
  • DOUBLE-STRANDED DNA
  • RUVC GENE-PRODUCT
  • ESCHERICHIA-COLI
  • CRYSTAL-STRUCTURE
  • SACCHAROMYCES-CEREVISIAE
  • BRANCH MIGRATION
  • 4-WAY JUNCTION

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