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

Junction-resolving enzymes are nucleases that are specific for the structur e 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 fluor escence anisotropy of Cy3 terminally attached to one of the helical arms of a four-way junction. By contrast, there was no change in fluorescein aniso tropy with the binding of single dimers of these proteins. Fluorescence res onance energy transfer has therefore been used between fluorescein and Cy3 fluorophores attached to the ends of helical arms to analyse the global str ucture of the junction on protein binding. The results indicate that both e nzymes induce a marked change in the global DNA conformation on the binding of a single dimer. The structure of the protein-junction complexes is inde pendent of the presence or absence of divalent metal ions, unlike that of t he protein-free junction. The structures of the RuvC and Hjc complexes are different, but both represent a significant opening of the structure compar ed 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 i n the function of these enzymes. (C) 2001 Academic Press.