Poster abstracts
Poster number 15 submitted by Motilal Uttarkabat
Dynamic nuclear polarization solid-state nuclear magnetic resonance reveals Hoogsteen base pairing in a DNA-Protein Complex
Motilal Uttarkabat (Biophysics Graduate Program), Serafima Guseva (Department of Biochemistry and Molecular Biophysics, Columbia University), Daniel W. Conroy (Department of Chemistry and Biochemistry, OSU), Hashim Al-Hashimi, Christopher Jaroniec (Biophysics Graduate Program, Department of Chemistry and Biochemistry, OSU)
Abstract:
Hoogsteen base pairs are alternative DNA conformations that are thermodynamically less stable than canonical Watson–Crick base pairs. They frequently occur in complexes with proteins and small molecules, where they play important roles in DNA recognition, repair, and replication [1]. Differentiating Hoogsteen from Watson–Crick base pairs in protein-bound DNA remains challenging for conventional structural biology techniques. These base pairs can be difficult to resolve by solution nuclear magnetic resonance (NMR) spectroscopy due to size limitations, by cryo-electron microscopy owing to limited resolution, and by X-ray crystallography because of ambiguous electron density. Here, we describe a solid-state NMR (ssNMR) method for characterizing Hoogsteen base pairs in protein-DNA complexes that relies on improved resolution from site-specific incorporation of 13C- and 15N-labeled nucleotides and enhanced signal from dynamic nuclear polarization (DNP) NMR [2]. This method was applied to resolve the conformation of an A–T base pair within a ~47 kDa complex formed between a 21-mer hairpin DNA and the IS608 transposase (TnPA) protein. In the free hairpin DNA, the A–T base pair adjacent to the tri-nucleotide apical loop exists in dynamic equilibrium with a minor Hoogsteen conformation (~14% population) stabilized by the loop residues [3]. The characteristic chemical shifts observed in the TnPA-bound DNA indicate that this base pair fully converts to A(syn)-T Hoogsteen geometry upon binding to the TnPA protein. Collectively, these results establish DNP enhanced ssNMR as a new and complementary strategy for resolving noncanonical DNA base pair conformations in protein-DNA complexes.
References:
1- Nikolova, Evgenia N., et al. "A historical account of hoogsteen base‐pairs in duplex DNA." Biopolymers 99.12 (2013): 955-968.
2- Conroy, Daniel W., et al. "Probing Watson-Crick and Hoogsteen base pairing in duplex DNA using dynamic nuclear polarization solid-state NMR spectroscopy." Proceedings of the National Academy of Sciences 119.30 (2022): e2200681119.
3- Guseva, Serafima, et al. "An A‐T Hoogsteen Base Pair in a Naked DNA Hairpin Motif: A Protein‐Recognized Conformation." Angewandte Chemie 137.25 (2025): e202425067.
Keywords: Protein-DNA Complex, NMR, Hoogsteen