Characterizing Watson-Crick versus Hoogsteen Base Pairing in a DNA-Protein Complex Using Nuclear Magnetic Resonance and Site-Specifically 13C- and 15N-Labeled DNA.
Journal Article (Journal Article)
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 nuclear magnetic resonance (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- and 15N-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 an ∼43 kDa complex between a 34 bp duplex DNA and the integration host factor (IHF) protein. In the crystal structure (Protein Data Bank entry 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 interproton nuclear Overhauser effects 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.
Full Text
Duke Authors
Cited Authors
- Zhou, H; Sathyamoorthy, B; Stelling, A; Xu, Y; Xue, Y; Pigli, YZ; Case, DA; Rice, PA; Al-Hashimi, HM
Published Date
- April 16, 2019
Published In
Volume / Issue
- 58 / 15
Start / End Page
- 1963 - 1974
PubMed ID
- 30950607
Pubmed Central ID
- PMC8091065
Electronic International Standard Serial Number (EISSN)
- 1520-4995
Digital Object Identifier (DOI)
- 10.1021/acs.biochem.9b00027
Language
- eng
Conference Location
- United States