The solution structure of the GC-rich non-self-complementary DNA 12-mer duplex (I), which contains a (+)-CC-1065 highly reactive bonding sequence 5′AGTTA* (where * denotes the [formula omitted] covalent modification site), has been examined thoroughly by one- and two-dimensional proton and phosphorus NMR spectroscopy, hydroxyl-radical footprinting, and NOESY restrained molecular mechanics and dynamics calculations. The assignments of the nonexchangeable proton resonances (except some of the H5′ and H5″ protons due to severe resonance overlap), phosphorus resonances, and the exchangeable resonances (except amino protons of adenosine and guanosine) of this 12-mer duplex have been made. The results show that this 12-mer duplex maintains an overall B-form DNA with all anti base orientation throughout in aqueous solution at room temperature. Hydroxyl-radical footprinting experiments on a 21-mer sequence that contains this 12-mer duplex used for NMR studies showed that the minor groove is somewhat narrowed at the 7G-8T and 17A-18C steps, as indicated by the inhibition of cleavage at these locations. Although both high-field NMR and hydroxyl-radical footprinting experiments supported a bent-like structure for this 12-mer duplex, nondenaturing gel electrophoresis on the ligated 21-mer sequence that contains this 12-mer duplex did not show the abnormally slow migration characteristic of a bent DNA duplex. Analysis of the NMR data sets reveals several local structural perturbations similar to those found on an (A)n tract DNA duplex. For example, the existence of a propeller twist was detected within the A·T-rich region for both the 12-mer and the (A)n tract DNA duplexes. The 18CH5 aromatic resonance that is directly adjacent to the 3′ side of the 5′TAA segment was significantly shifted upfield with a chemical shift of 5.10 ppm, which is almost within the region normally associated with sugar H3′ protons. The sugar geometries for 18C and 7G, which are located to the 3′ side of the 5′TAA segment, are proposed to be in the neighborhood of C3′-endo and O1′-endo ⟺ C3′-endo, respectively. We propose that this unusually upfield-shifted resonance signal for 18CH5 and the average C3′-endo sugar geometry for 18C nucleotide on the 12-mer duplex is connected with the peculiar conformation, possibly a transient kink, within the 5′AC/GT step. The results of the NOESY restrained molecular mechanics and dynamics calculations on the 12-mer sequence reveal two kinks, which are located on either side of the 18C nucleotide that has an average C3′-endo sugar geometry. The two phosphorus resonance signals that are located at the 7G-8T and the 18C-19T steps, where the minor groove is narrowed as indicated by the hydroxyl-radical footprinting experiments, displayed unusual upfield chemical shifts. Also identified were two unusually broadened base protons of the 16A nucleotide and one imino proton belonging to the 9T-16A base pair within the A·T-rich segment. We proposed that this broadening phenomenon is most likely due to a unique internal motion characterized by a rapid local conformational equilibrium between microstates of the 12-mer duplex in aqueous solution at room temperature. This local conformational flexibility, a transient kink, and the bent-like structure are proposed to play a critical role in the sequence-specific recognition of the DNA duplex by (+)-CC-1065.
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