NMR spectroscopic and enzymatic studies of DNA hairpins containing mismatches in the EcoRI recognition site

We have correlated the structural perturbations caused by DNA mismatches wi th the enzymatic data of the interaction of the restriction endonuclease Ec oRI with DNA. Oligonucleotides d(CGAGAATTCTCA(5)GAXAATTCT) (X = G, A, T) an d d(CGCGAATTYGCGT(4)CGCXAATTCGCG) (Y = C, X = G, T and Y = A, X = T) contai ning single mismatches within the EcoRI recognition site were characterized by NMR spectroscopy and by their EcoRI substrate properties. UV melting an d gel electrophoresis studies confirm that the oligonucleotides form hairpi n structures. The presence of either a CT or a CA mismatch results in marke dly lower T-m and van't Hoff enthalpies compared with the fully base paired control. NMR imino proton spectra of these hairpins demonstrate that the p erturbation caused by the two mispairs or a noncanonical AT pair is localiz ed and limited to one or two base pairs on either side of the perturbation. The DNA hairpin structures containing single mismatches, and to a lesser e xtent also sequences with a single noncanonical base pair, an substrates fo r the restriction endonuclease. In addition to the strand scission at the n onperturbed GpA phosphodiester bond some cleavage is observed at the mismat ched position. The interactions of the CA and CT mismatched hairpin with th e enzyme are characterized by binding constants that are only 33 and 57 tim es lower, respectively, than that for the canonical sequence, corresponding to 8-10 kJ.mol(-1) less favorable free binding energy. This, taken togethe r with the NMR data, indicates that the CA and CT mismatches have only smal l effects on the EcoRI recognition of the DNA substrate. We conclude that t wo out of the three hydrogen bonds that characterize the interaction of Eco RI with the CG base pair in the canonical sequence can still be formed for either the CT or CA mismatched recognition site.