Refinement of the solution structure of the DNA dodecamer 5'd(CGCGPATTCGCG)2 containing a stable purine-thymine base pair: combined use of nuclear magnetic resonance and restrained molecular dynamics

Abstract

The solution structure of the self-complementary dodecamer 5'd(CGCGPATTCGCG)2, containing a purine-thymine base pair within the hexameric canonical recognition site GAATTC for the restriction endonuclease EcoRI, is investigated by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Nonexchangeable and exchangeable protons are assigned in a sequential manner. A set of 228 approximate interproton distance restraints are derived from two-dimensional nuclear Overhauser enhancement spectra recorded at short mixing times. These distances are used as the basis for refinement using restrained molecular dynamics in which the interproton distance restraints are incorporated into the total energy function of the system in the form of effective potentials. Eight calculations are carried out, four starting from classical A-DNA and four from classical B-DNA. In all cases convergence to very similar B-type structures is achieved with an average atomic root mean square (rms) difference between the eight converged structures of 0.7 +/- 0.2 A, compared to a value of 6.5 A for that between the two starting structures. It is shown that the introduction of the purine-thymine mismatch does not result in any significant distortion of the structure. The variations in the helical parameters display a clear sequence dependence. The variation in helix twist and propeller twist follows Calladine's rules and can be attributed to the relief of interstrand purine-purine clash at adjacent base pairs. Overall the structure is straight. Closer examination, however, reveals that the central 5 base pair steps describe a smooth bend directed toward the major groove with a radius of curvature of approximately 38 A, which is compensated by two smaller kinks in the direction of the minor groove at base pair steps 3 and 9. These features can be explained in terms of the observed variation in roll and slide.

Effects of functional group changes in the EcoRV recognition site on the cleavage reaction catalyzed by the endonuclease

“…This is in agreement with the reported preliminary studies involving similar modifications (Fliess et al, 1986;Nwosu et al, 1988). The high-resolution NMR-derived structure of an oligodeoxynucleotide containing a purinethymine base pair indicates that, although an interstrand hydrogen bond is missing, the purine occupies the same position in the helm and its orientation with respect to thymine is the same as for a native adenine-thymine base pair (Clore et al, 1988). On the basis of these observations, it is unlikely that the lack of substrate activity observed with the deletion of either amino group is a function of a significant structural aberration induced by the presence of the purine base.…”

The Paperclip Triplex: Understanding the Role of Apex Residues in Tight Turns

“…Resonance assignments were accomplished by the NOE-observable 1 H-1 H distances between neighboring nucleotides in the sequence (44)(45)(46)(47).…”

Solution structure of a pyrimidine·purine·pyrimidine DNA triplex containing T·AT, C+ ·GC and G·TA triples

“…Taking spin-diffusion effects and accuracy of the quantitation protocol into consideration, the intensities and cross relaxation rates derived from nuclear Overhauser enhancement spectra (NOESY) [54] and NOESY-total correlation spectroscopy spectra (NOESY-TOCSY) for the various proton pairs were translated into distances and assigned bounds (see Materials and methods). Distances from the three-dimensional spectrum were obtained by comparing the intensities of cross peaks resulting from Hx-NOE-H2' (H2")-J-+H1' with the intensities of cross peaks resulting from H2" (H2')--NOE-+H2' (H2")-J-*H1', and by comparing Hy --NOEH6-JH5 magnetization transfers with HS--NoE-H6-J-H5 transfers (Hx and Hy represent any DNA proton) [52].…”

“…for which the JHI'-H2' and JH1'-H2" coupling constants implicated them in S-type or N-type sugar puckers [54,55].…”