title: 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 ColE1 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(1,10-phenanthroline)ruthenium(II), Ru(phen)3(2+), tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II), Ru(DIP)3(2+), and tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), Co(DIP)3(3+), in general, bind stereoselectively to DNA helices, with enantiomers possessing the delta 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 lambda enantiomer, which binds only poorly to a B-DNA helix, that inhibits EcoRI preferentially. Kinetic studies in the presence of lambda-Ru(DIP)3(2+) indicate that the enzyme inhibition occurs as a result of the lambda 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 lambda-Co(DIP)3(3+), until a protein concentration is reached where specific DNA recognition sites are saturated with enzyme. Thus, although lambda-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 lambda 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.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI; D004926:Escherichia coli / Q000235:genetics; D007364:Intercalating Agents / Q000494:pharmacology*; D007700:Kinetics; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D007364:Intercalating Agents; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI