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

Abstract

We have correlated the structural perturbations caused by DNA mismatches with the enzymatic data of the interaction of the restriction endonuclease EcoRI with DNA. Oligonucleotides d(CGAGAATTCTCA5GAXAATTCT) (X = G, A, T) and d(CGCGAATTYGCGT4CGCXAATTCGCG) (Y = C, X = G, T and Y = A, X = T) containing single mismatches within the EcoRI recognition site were characterized by NMR spectroscopy and by their EcoRI substrate properties. UV melting and gel electrophoresis studies confirm that the oligonucleotides form hairpin structures. The presence of either a CT or a CA mismatch results in markedly lower Tm and van't Hoff enthalpies compared with the fully base paired control. NMR imino proton spectra of these hairpins demonstrate that the perturbation caused by the two mispairs or a noncanonical AT pair is localized 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 extent also sequences with a single noncanonical base pair, are substrates for the restriction endonuclease. In addition to the strand scission at the nonperturbed GpA phosphodiester bond some cleavage is observed at the mismatched position. The interactions of the CA and CT mismatched hairpin with the enzyme are characterized by binding constants that are only 33 and 57 times lower, respectively, than that for the canonical sequence, corresponding to 8-10 kJ·mol-1 less favorable free binding energy. This, taken together with the NMR data, indicates that the CA and CT mismatches have only small effects on the EcoRI recognition of the DNA substrate. We conclude that two out of the three hydrogen bonds that characterize the interaction of EcoRI with the CG base pair in the canonical sequence can still be formed for either the CT or CA mismatched recognition site.Key words: DNA hairpins, EcoRI recognition, mismatches, imino protons.