CONFORMATIONAL FLEXIBILITY IN DNA DUPLEXES CONTAINING SINGLE G-CENTER-DOT-G MISMATCHES

Purine-purine mismatches can base-pair in a variety of configurations depending on solution conditions. The G . G mismatch, which also occurs in the G-quartet structure, has been showm by both x-ray crystallography and NMR to adopt G(anti) . G(syn) mispairs, with very different hydrogen bonding patterns [Skelly, J., Edwards, K., Jenkins, T. C. and Neidle, S. (1993) Proc. Natl Acad Sci. USA 90, 804-8O8; Cognet, J. A. H., Gabarro-Arpa, J., Le Bret, M., van der Marel, G. A. van Boom, J. H. and Fazakerley, G. V. (1991) Nucleic Acids. Res. 19, 6771-6779] while we have recently suggested the presence of weakly hydrogen-bonded G(anti) . G(anti) pairs in solution [Borden, K. L. B., Jenkins, T. C., Skelly, J. V., Brown, T. and Lane, A. N. (1992) Biochemistry 31, 5411-5422]. Spectral overlap and additional exchange processes have made detailed structural analysis difficult in these mismatched oligomers. We have used NMR to characterise the conformations of four duplexes containing single G . G mismatches, including st nonamer d(CATCGGATG), two undecamers d(GCATTGAATGC) and d(CATGTGACGTG) that can each form a self-complementary duplex with a single G . G mispair in the centre, and the nonself-complementary d(GTAACGACATG) . d(CATGTGGTTAC). The three self-complementary duplexes have a single set of NMR resonances, and all four duplexes show evidence of conformational exchange at the mismatch site. The N1H resonances of the mismatched G residues each integrate to two protons, ruling out the enol tautomer. They resonate between 10.5-10.7 ppm, far upfield of the Watson-Crick hydrogen-bonded GN1H and exchange readily with water protons. Intraresidue GH8-H1' NOE intensities are two-threefold larger for the mismatched G residues than in G . C base pairs, indicating the presence of syn conformations. NOE time courses for the self-complementary duplexes were consistent with an equimolar mixture of G(syn) . G(anti) and G (anti) . G(syn) states. By symmetry, these states must be interconverting at a rate that is fast on the chemical shift timescale. In the non-self-complementary undecamer, the NOE data indicated that the distinguishable mismatched G residues also spend a significant, but different, fraction of the time in both the syn and anti conformations. The rate constant for the syn/anti transition in the non-self-complementary undecamer was determined as approximate to 14000 s(-1) at 303 K from rotating frame T-1 measurements, and the apparent frequency difference was > 250 Hz. Calculations based on NOEs and coupling constants showed that the duplexes are overall in the B form. Improved agreement with the NOE data for the mismatched residues could be obtained by constructing linear averages of conformations for the mismatched bases.