SEQUENCE DEPENDENCE AND DIRECT MEASUREMENT OF CROSSOVER ISOMER DISTRIBUTION IN MODEL HOLLIDAY JUNCTIONS USING NMR-SPECTROSCOPY

A 32-base-pair model of the Holliday junction (HJ) intermediate in gen etic recombination has been prepared and analyzed in-depth by 2D and 3 D H-1 NMR spectroscopy. This HJ (J2P1) corresponds to a cyclic permuta tion of the base pairs at the junction relative to a previously studie d HJ [J2; Chen, S.-M., & Chazin, W.J. (1994) Biochemistry 33, 11453-11 459], designed to probe the effect of the sequence at the n - 1 positi on (where n is the residue directly at the branch point) on the stacki ng geometry. Observation of several interbase nuclear Overhauser effec ts (NOEs) clearly indicates a strong preference for the isomer opposit e that observed for J2, confirming the dependence of stacking isomer p reference on the sequence at the junction. As for other model HJs stud ied, a small equilibrium distribution of the alternate isomer could be identified. A sample of J2P1 was prepared with a single N-15-labeled thymine residue at the branch point. 1D N-15-filtered H-1-detected exp eriments on this sample at low temperature give strong support for the co-existence of the two stacking isomers and provide a much more dire ct and accurate measure of the crossover isomer distribution. The comp arative analysis of our immobile HJs and a model cruciform structure [ Pikkemaat, J.A., van den Elst, H., van Boom, J.H., & Altona, C. (1994) Biochemistry 33, 14896-14907] sheds new light on the issue of the rel evance of crossover isomer preference in vivo.