STRUCTURAL SIMILARITIES AND DIFFERENCES BETWEEN H1-FAMILY AND H2-FAMILY DNA MINIHAIRPIN LOOPS - NMR-STUDIES OF OCTAMERIC MINIHAIRPINS

The DNA sequences 5'-d(CGC-AC-GCG)-3' (HPAC), 5'-d(CGC-AA-GCG)-3' (HPA A), 5'-d(CGC-TC-GCG)-3' (HPTC) and 5'-d(CGC-CT-GCG)-3' (HPCT), were st udied by means of nmr spectroscopy. At low DNA concentration and no ad ded salt all four molecules adapt a minihairpin structure, containing three Watson-Crick base pairs and a two-residue loop. The structure of the HPAC hairpin is based on quantitative distance restraints, derive d by a full relaxation matrix approach (iterative relaxation matrix ap proach), together with torsion angles obtained from coupling constant analysis. The loop folding is of the H1-family type, characterized by continuous 3'-5' stacking of the loop bases on the duplex stem. The st ructure of the HPAA hairpin is similar to that of HPAC, but is more fl exible and has a lower thermodynamic stability (T-m 326 K vs 320 K). A ccording to ''weakly'' distance-constrained simulations in water on th e HPAC minihairpin, the typical H1-family loop folding remains intact during the simulation. However; residue-based R factors of simulated n uclear Overhauser effect spectroscopy spectra, free molecular dynamics simulations in vacuo, and unusual chemical shift profiles indicate pa rtial destacking of the loop bases at temperatures below the overall m elting midpoint The dynamic nature of the loop bases gives insight int o the geometrical tolerances of stacking between bases in H1-family mi nihairpin loops. The HPTC and HPCT minihairpins, both containing a pyr imidine base at the first position in the loop, adopt a H2-family type folding, in which the first loop base is loosely bound in the minor g roove and the second loop base is stacked upon the helix stem. The the rmal stability for these two hairpins corresponds to 327-329 K, but de pends on local base sequence. Preference for the type of folding depen ds on a single substitution from a pyrimidine (H2 family) to a purine (H2 family) at the first position of the miniloop and is explained by differences in base stacking energies, steric size, and the number of possible candidates for hydrogen bonds in the minor groove. In view of newly collected data, previous models of the H1-family and H2-family hairpins had to be revised and are now compatible with the reported HP TC and HPAC structures. The structural difference between the refined structure of HPAC and HPTC show that a conversion between H1-family an d H2-family hairpins is geometrically possible by a simple pivot point rotation of 270 degrees H1-family folding toward a position in the mi nor groove in a H2-family folding. The second loop residue subsequentl y shifts to the position of the first base in a concerted fashion. (C) 1998 John Wiley & Sons, Inc.