RESUMEN
The propensities to form lowly-populated short-lived conformations of DNA could vary with sequence, providing an important source of sequence-specificity in biochemical reactions. However, comprehensively measuring how these dynamics vary with sequence is challenging. Using 1H CEST and 13C R1ρ NMR, we measured Watson-Crick to Hoogsteen dynamics for an A-T base pair in thirteen trinucleotide sequence contexts. The Hoogsteen population and exchange rate varied 4-fold and 16-fold, respectively, and were dependent on both the 3'- and 5'-neighbors but only weakly dependent on monovalent ion concentration (25 versus 100 mM NaCl) and pH (6.8 versus 8.0). Flexible TA and CA dinucleotide steps exhibited the highest Hoogsteen populations, and their kinetics rates strongly depended on the 3'-neighbor. In contrast, the stiffer AA and GA steps had the lowest Hoogsteen population, and their kinetics were weakly dependent on the 3'-neighbor. The Hoogsteen lifetime was especially short when G-C neighbors flanked the A-T base pair. The Hoogsteen dynamics had a distinct sequence-dependence compared to duplex stability and minor groove width. Thus, our results uncover a unique source of sequence-specificity hidden within the DNA double helix in the form of A-T Hoogsteen dynamics and establish the utility of 1H CEST to quantitively measure sequence-dependent DNA dynamics.
RESUMEN
Many biochemical processes use the Watson-Crick geometry to distinguish correct from incorrect base pairing. However, on rare occasions, mismatches such as G·T/U can transiently adopt Watson-Crick-like conformations through tautomerization or ionization of the bases, giving rise to replicative and translational errors. The propensities to form Watson-Crick-like mismatches in RNA:DNA hybrids remain unknown, making it unclear whether they can also contribute to errors during processes such as transcription and CRISPR/Cas editing. Here, using NMR R1ρ experiments, we show that dG·rU and dT·rG mismatches in two RNA:DNA hybrids transiently form tautomeric (Genol·T/U $ \mathbin{\lower.3ex\hbox{$\buildrel\textstyle\rightarrow\over {\smash{\leftarrow}\vphantom{_{\vbox to.5ex{\vss}}}}$}}$ G·Tenol/Uenol) and anionic (G·T-/U-) Watson-Crick-like conformations. The tautomerization dynamics were like those measured in A-RNA and B-DNA duplexes. However, anionic dG·rU- formed with a ten-fold higher propensity relative to dT-·rG and dG·dT- and this could be attributed to the lower pKa (ΔpKa â¼0.4-0.9) of U versus T. Our findings suggest plausible roles for Watson-Crick-like G·T/U mismatches in transcriptional errors and CRISPR/Cas9 off-target gene editing, uncover a crucial difference between the chemical dynamics of G·U versus G·T, and indicate that anionic Watson-Crick-like G·U- could play a significant role evading Watson-Crick fidelity checkpoints in RNA:DNA hybrids and RNA duplexes.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Hibridación de Ácido Nucleico , Emparejamiento Base , ADN/genética , ADN/química , Conformación de Ácido Nucleico , ARN/químicaRESUMEN
Many biochemical processes use the Watson-Crick geometry to distinguish correct from incorrect base pairing. However, on rare occasions, mismatches such as Gâ¢T/U can transiently adopt Watson-Crick-like conformations through tautomerization or ionization of the bases, giving rise to replicative and translational errors. The propensities to form Watson-Crick-like mismatches in RNA:DNA hybrids remain unknown, making it unclear whether they can also contribute to errors during processes such as transcription and CRISPR/Cas editing. Here, using NMR R 1ρ experiments, we show that dGâ¢rU and dTâ¢rG mismatches in two RNA:DNA hybrids transiently form tautomeric (G enol â¢T/U âGâ¢T enol /U enol ) and anionic (Gâ¢T - /U - ) Watson-Crick-like conformations. The tautomerization dynamics were like those measured in A-RNA and B-DNA duplexes. However, anionic dGâ¢rU - formed with a ten-fold higher propensity relative to dT - â¢rG and dGâ¢dT - and this could be attributed to the lower pK a (Δ pK a â¼0.4-0.9) of U versus T. Our findings suggest plausible roles for Watson-Crick-like Gâ¢T/U mismatches in transcriptional errors and CRISPR/Cas9 off-target gene editing, uncover a crucial difference between the chemical dynamics of Gâ¢U versus Gâ¢T, and indicate that anionic Watson-Crick-like Gâ¢U - could play a significant role evading Watson-Crick fidelity checkpoints in RNA:DNA hybrids and RNA duplexes.
RESUMEN
Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes1. These interactions are mediated by intermolecular contacts, which if disrupted, lead to alterations in cell physiology. Nevertheless, the formation of intermolecular contacts nearly universally requires changes in the conformations of the interacting biomolecules. As a result, binding affinity and cellular activity crucially depend both on the strength of the contacts and on the inherent propensities to form binding-competent conformational states2,3. Thus, conformational penalties are ubiquitous in biology and must be known in order to quantitatively model binding energetics for protein and nucleic acid interactions4,5. However, conceptual and technological limitations have hindered our ability to dissect and quantitatively measure how conformational propensities affect cellular activity. Here we systematically altered and determined the propensities for forming the protein-bound conformation of HIV-1 TAR RNA. These propensities quantitatively predicted the binding affinities of TAR to the RNA-binding region of the Tat protein and predicted the extent of HIV-1 Tat-dependent transactivation in cells. Our results establish the role of ensemble-based conformational propensities in cellular activity and reveal an example of a cellular process driven by an exceptionally rare and short-lived RNA conformational state.
Asunto(s)
Duplicado del Terminal Largo de VIH , VIH-1 , Conformación de Ácido Nucleico , ARN Viral , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Duplicado del Terminal Largo de VIH/genética , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/genética , VIH-1/metabolismoRESUMEN
Replicative errors contribute to the genetic diversity needed for evolution but in high frequency can lead to genomic instability. Here, we show that DNA dynamics determine the frequency of misincorporating the Aâ¢G mismatch, and altered dynamics explain the high frequency of 8-oxoguanine (8OG) Aâ¢8OG misincorporation. NMR measurements revealed that Aantiâ¢Ganti (population (pop.) of >91%) transiently forms sparsely populated and short-lived Aanti+â¢Gsyn (pop. of ~2% and kex = kforward + kreverse of ~137 s-1) and Asynâ¢Ganti (pop. of ~6% and kex of ~2,200 s-1) Hoogsteen conformations. 8OG redistributed the ensemble, rendering Aantiâ¢8OGsyn the dominant state. A kinetic model in which Aanti+â¢Gsyn is misincorporated quantitatively predicted the dAâ¢dGTP misincorporation kinetics by human polymerase ß, the pH dependence of misincorporation and the impact of the 8OG lesion. Thus, 8OG increases replicative errors relative to G because oxidation of guanine redistributes the ensemble in favor of the mutagenic Aantiâ¢8OGsyn Hoogsteen state, which exists transiently and in low abundance in the Aâ¢G mismatch.
Asunto(s)
Daño del ADN , ADN , Humanos , Emparejamiento Base , ADN/química , MutagénesisRESUMEN
As the Watson-Crick faces of nucleobases are protected in dsDNA, it is commonly assumed that deleterious alkylation damage to the Watson-Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson-Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to ssDNA has not been quantified. Watson-Crick base pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base pairs that expose the Watson-Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson-Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson-Crick A-T base pairs in dsDNA only conferred â¼130-fold protection against adenine-N1 methylation, and this protection was reduced to â¼40-fold for A(syn)-T Hoogsteen base pairs embedded in a DNA-drug complex. Our results indicate that Watson-Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-wide in vivo.