Restriction Endonuclease EcoRI Alters the Enantiomeric Preference of Chiral Metallointercalators for DNA: An Illustration of a Protein-Induced DNA Conformational Change

Abstract

A conformational change in the DNA plasmid ColE_1 appears to occur upon specific binding of the restriction endonuclease EcoRI. Enzyme association alters the chiral discrimination found in binding metallointercalators to DNA sites. The complexes tris(l,10-henanthroline)ruthenium(II), Ru(phen)_3^(2+), tris( 4,7-diphenyl-1, 10-phenanthroline) ruthenium(II), Ru(DIP)_3^(2+), and tris( 4,7-diphenyl- l, 10-phenanthroline)cobalt(III), Co(DIP)_3^(3+), in general, bind stereoselectively to DNA helices, with enantiomers possessing the Δ configuration bound preferentially by right-handed B-DNA. In the presence of EcoRI, however, this enantioselectivity is altered. The chiral intercalators, at micromolar concentrations, inhibit the reaction of EcoRI, but for each enantiomeric pair it is the Λ enantiomer, which binds only poorly to a B-DNA helix, that inhibits EcoRI preferentially. Kinetic studies in the presence of Λ-Ru(DIP)_3^(2+) indicate that the enzyme inhibition occurs as a result of the Λ enantiomer binding to the enzyme-DNA complex as well as to the free enzyme. Furthermore, photolytic strand cleavage experiments using Co(DIP)_3^(3+) indicate that the metal complex interacts directly at the protein-bound DNA site. Increasing concentrations of bound EcoRI stimulate photoactivated cleavage of the DNA helix by Λ-Co(DIP)_3^(3+), until a protein concentration is reached where specific DNA recognition sites are saturated with enzyme. Thus, although Λ-Co(DIP)_3^(3+) does not bind closely to the DNA in the absence of enzyme, specific binding of EcoRI appears to alter the DNA structure so as to permit the close association of the Λ isomer to the DNA helix. Mapping experiments demonstrate that this association leads to photocleavage of DNA by the cobalt complex at or very close to the EcoRI recognition site. This study provides evidence that in solution, under enzymatic conditions, a DNA-binding protein may distort the DNA helical structure and furthet illustrates how small molecular probes of DNA conformation might be used in examining the structure of protein-bound DNA sites.