STRUCTURAL EFFECTS OF THE C2-METHYLHYPOXANTHINE-CYTOSINE BASE-PAIR INB-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 structu ral consequence of the m2I incorporation in DNA by a combination of X- ray crystallographic, NMR, and enzymatic analyses. The crystal structu re of d(CGC[m2I]AATTCGCG) has been solved and refined to an R factor o f 20.7% at 2.25-angstrom resolution. In the DNA duplex, the two indepe ndent 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 bas e-paired cytosine, it only causes the base pair to have slightly highe r propeller twist and buckle angles. Its solution structure was analyz ed by the NMR refinement procedure SPEDREF [Robinson, H., & Wang, A. H .-J. (1992) Biochemistry 31, 3524-3533] using 2D nuclear Overhauser ef fect data. Two starting models, a relaxed fiber model and an X-ray mod el, were subjected to the NOE-constrained refinement using 1518 NOE cr oss-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 mod els (all atoms included) is 1.23 angstrom, which presently seems to be near the limit of convergence of NOE-based refinement. The local stru ctures of the two models are in better agreement as measured by the RM SD of the dinucleotide steps, falling in the range 0.54-0.98 angstrom. Both refined solution structures confirm that the m2I dodecamer struc ture is of the B-DNA type with a narrow minor groove at the AT region, as observed in the crystal. However, significant differences exist be tween the crystal and solution structures in parameters such as pseudo rotation angles, propeller twist angles, etc. The solution structure t ends to have a more uniform backbone conformation, an observation cons istent with that concluded from the laser Raman study of d(CGCAAATTTGC G) [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 rel ated dodecamers, d(CGCGAATTCGCG), d(CGC[m2I]AATTCGCG), and d(CGC[e6G]A ATTCGCG), were tested as substrates for the restriction endonuclease E coRI. The m2I dodecamer was active, but the e6G dodecamer was not. Our results illustrate the complementarity in terms of the structural inf ormation provided by the two methods, X-ray diffraction and NMR.