Probing conformational transitions towards mutagenic Watsonâ??Crick-like G·T mismatches using off-resonance sugar carbon R 1Ď? relaxation dispersion
Abstract
NMR off-resonance
R1Ď? relaxation dispersion measurements on base carbon and nitrogen nuclei have revealed that wobble G·T/U mismatches in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-abundance, and mutagenic Watsonâ??Crick-like conformations. As Watsonâ??Crick-like G·T mismatches have base pairing geometries similar to Watsonâ??Crick base pairs, we hypothesized that they would mimic Watsonâ??Crick base pairs with respect to the sugar-backbone conformation as well. Using off-resonance
R1Ď? measurements targeting the sugar C3â?˛ and C4â?˛ nuclei, a structure survey, and molecular dynamics simulations, we show that wobble G·T mismatches adopt sugar-backbone conformations that deviate from the canonical Watsonâ??Crick conformation and that transitions toward tautomeric and anionic Watsonâ??Crick-like G·T mismatches restore the canonical Watsonâ??Crick sugar-backbone. These measurements also reveal kinetic isotope effects for tautomerization in D2O versus H2O, which provide experimental evidence in support of a transition state involving proton transfer. The results provide additional evidence in support of mutagenic Watsonâ??Crick-like G·T mismatches, help rule out alternative inverted wobble conformations in the case of anionic G·Tâ??, and also establish sugar carbons as new non-exchangeable probes of this exchange process.
Source: Journal of Biomolecular NMR