Structural Effects of the C²-Methylhypoxanthine: Cytosine Base Pair in B-DNA: A Combined NMR and X-ray Diffraction Study of d(CGC[m²I]AATTCGCG)

C²-Methylhypoxanthine (m²I) is a synthetic analog of guanine with the N²-amino group replaced by a methyl group. We have studied the structural consequence of the m²I incorporation in DNA by a combination of X-ray crystallographic, NMR, and enzymatic analyses. The crystal structure of d(CGC-[m²I] AATTCGCG) has been solved and refined to an R factor of 20.7% at 2.25-Å resolution. In the DNA duplex, the two independent m²I:C base pairs maintain the Watson-Crick scheme. While the C²-methyl group of m²I is in van der Waals contact with the O² of the base-paired cytosine, it only causes the base pair to have slightly higher propeller twist and buckle angles. Its solution structure was analyzed by the NMR refinement procedure SPEDREF [Robinson, H., & Wang, A.H.-J. (1992) Biochemistry 31, 3524–3533] using 2D nuclear Overhauser effect data. Two starting models, a relaxed fiber model and an X-ray model, were subjected to the NOE-constrained refinement using 1518 NOE cross-peak integrals to arrive at the final models with (NOE) R factors of 13.8% and 14.3%, respectively. The RMSD between the two refined models (all atoms included) is 1.23 Å, which presently seems to be near the limit of convergence of NOE-based refinement. The local structures of the two models are in better agreement as measured by the RMSD of the dinucleotide steps, falling in the range 0.54–0.98 Å. Both refined solution structures confirm that the m²I dodecamer structure is of the B-DNA type with a narrow minor groove at the AT region, as observed in the crystal. However, significant differences exist between the crystal and solution structures in parameters such as pseudorotation angles, propeller twist angles, etc. The solution structure tends to have a more uniform backbone conformation, an observation consistent with that concluded from the laser Raman study of d (CGCAAATTTGCG) [Benevides, J. M., Wang, A.H.-J., van der Marel, G. A., van Boom, J. H., & Thomas, G. J., J. (1988) Biochemistry 27, 931–938]. Three related dodecamers, d(CGCGAATTCGCG), d(CGC[m²I]AATTCGCG), and d(CGC[e⁶G]AATTCGCG), were tested as substrates for the restriction endonuclease EcoRI. The m²I dodecamer was active, but the e⁶G dodecamer was not. Our results illustrate the complementarity in terms of the structural information provided by the two methods, X-ray diffraction and NMR.