Characterizing Watson-Crick versus Hoogsteen base-pairing in a DNA-protein complex using NMR and site-specifically 13C/15N labeled DNA

A( syn )-T and G( syn )-C + Hoogsteen base pairs in protein-bound DNA duplexes can be difficult to resolve by X-ray crystallography due to ambiguous electron density and by NMR spectroscopy due to poor chemical shift dispersion and size limitations with solution-state NMR spectroscopy. Here we describe an NMR strategy for characterizing Hoogsteen base pairs in protein-DNA complexes, which relies on site-specifically incorporating 13C/ 15 N labeled nucleotides into DNA duplexes for unambiguous resonance assignment and to improve spectral resolution. The approach was used to resolve the conformation of an A-T base pair in a crystal structure of a ~43 kD complex between a 34-bp duplex DNA and the integration host factor (IHF) protein. In the crystal structure (PDB: 1IHF), this base pair adopts an unusual Hoogsteen conformation with a distorted sugar-backbone that is accommodated by a nearby nick used to aid in crystallization. The NMR chemical shifts and inter-proton NOEs indicate that this base pair predominantly adopts a Watson-Crick conformation in the intact DNA-IHF complex under solution conditions. Consistent with these NMR findings, substitution of 7-deazaadenine at this base pair resulted in only a small (~2-fold) decrease in the IHF-DNA binding affinity. The NMR strategy provides a new approach for resolving crystallographic ambiguity and more generally for studying the structure and dynamics of protein-DNA complexes in solution.