Visualizing transient Watson–Crick-like mispairs in DNA and RNA duplexes

Abstract

Rare tautomeric and anionic nucleobases are believed to play fundamental biological roles but their prevalence and functional importance has remained elusive because they exist transiently, in low-abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-populated Watson-Crick like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10−3-10−5) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases.

Dynamic basis for dG•dT misincorporation via tautomerization and ionization

“…2a). Experiments were carried out at near-neutral pH (6.4–6.9) so as to lower the anionic ES2 below detection 8 (Extended Data Fig. 2b).…”

“…2c and Supplementary Tables 2,3) 8 . The tautomeric equilibria (Supplementary Table 2) obtained from this analysis and from re-fitting the RD data using a 3-state model with linear topology 25 (wobble⇌G•T enol /U enol ⇌G enol •T/U) are slightly tilted in favor of dG enol •dT in DNA ( K t = 2.1-4.6) whereas the populations of rG enol •rU and rG•rU enol are more comparable in RNA ( K t = 0.5-1.1).…”

“…While it remains unclear which WC-like mismatch contributes to replication and translation errors, factors (e.g. changes in pH 7,8,14,18 and chemical modifications 19 ) that stabilize different forms have been shown to increase misincorporation probabilities 14,20 . Misincorporation probabilities can also vary significantly with sequence context through mechanisms that remain poorly understood 21,22 .…”

“…Using NMR RD, we recently provided evidence that wobble G•T/U mismatches exist in dynamic equilibrium with tautomeric (ES1) and anionic (ES2) WC-like mismatches within DNA and RNA duplexes 8,31 . The guanine N1 (G-N1) and thymidine/uridine N3 (T/U-N3) chemical shifts measured for tautomeric ES1 were consistent with G enol •T/U, but were partially skewed toward G•T enol /U enol .…”

“…The guanine N1 (G-N1) and thymidine/uridine N3 (T/U-N3) chemical shifts measured for tautomeric ES1 were consistent with G enol •T/U, but were partially skewed toward G•T enol /U enol . This was interpreted as evidence for a rapid (on the chemical shift timescale) equilibrium between a major G enol •T/U and minor G•T enol /U enol species 8 . The anionic ES2 was only detectable at high pH (≥7.8) and was heavily skewed in favor of G•T − /U − with no evidence for G − •T/U.…”