Structural similarities and differences between H1- and H2-family DNA minihairpin loops: NMR studies of octameric minihairpins

Abstract

TheDNA sequences 5′-d(CGC-AC-GCG)-3′ (HPAC), 5′-d(CGC-AA-GCG)-3′ (HPAA), 5′-d(CGC-TC-GCG)-3′ (HPTC), and 5′-d(CGC-CT-GCG)-3′ (HPCT), were studied by means of nmr spectroscopy. At low DNA concentration and no added salt all four molecules adopt 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, derived by a full relaxation matrix approach (iterative relaxation matrix approach), 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 structure of the HPAA hairpin is similar to that of HPAC, but is more flexible and has a lower thermodynamic stability (Tm 326 K vs 320 K). According to “weakly” distance-constrained simulations in water on the HPAC minihairpin, the typical H1-family loop folding remains intact during the simulation. However, residue-based R factors of simulated nuclear Overhauser effect spectroscopy spectra, free molecular dynamics simulations in vacuo, and unusual chemical shift profiles indicate partial destacking of the loop bases at temperatures below the overall melting midpoint. The dynamic nature of the loop bases gives insight into the geometrical tolerances of stacking between bases in H1-family minihairpin loops. The HPTC and HPCT minihairpins, both containing a pyrimidine 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 groove and the second loop base is stacked upon the helix stem. The thermal stability for these two hairpins corresponds to 327–329 K, but depends on local base sequence. Preference for the type of folding depends on a single substitution from a pyrimidine (H2 family) to a purine (H1 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 HPTC and HPAC structures. The structural difference between the refined structure of HPAC and HPTC show that a conversion between H1-family and H2-family hairpins is geometrically possible by a simple pivot point rotation of 270° along two torsion angles, thereby swiveling the first loop base from a stacked position in a H1-family folding toward a position in the minor groove in a H2-family folding. The second loop residue subsequently shifts to the position of the first base in a concerted fashion. © 1998 John Wiley & Sons, Inc. Biopoly 46: 375–393, 1998