Biology subjects

Guseva, S.

Publications and source records attributed to Guseva, S..

4 recordsLinked to original sources

bioRxiv2024

An A-T Hoogsteen base pair in a naked DNA hairpin motif: A Protein-Recognized Conformation

In duplex DNA, A-T and G-C form Watson-Crick base pairs, and Hoogsteen pairing only dominates upon protein binding or DNA damage. Using NMR, we show that an A-T Hoogsteen base pair previously observed in crystal structures of transposon DNA hairpins bound to TnpA protein forms in solution even in the absence of TnpA. This Hoogsteen base pair, located adjacent to a dinucleotide apical loop, exists in dynamic equilibrium with a minor Watson-Crick conformation (population [~]11% and lifetime [~]55 {micro}s). Extending the apical loop to three residues inverted the equilibrium, making Watson-Crick the dominant state and the Hoogsteen conformation recognized by TnpA a minor state (population [~]14% and lifetime [~]28 {micro}s). The propensity for Hoogsteen pairing depended on apical loop residues, which form contacts directly or indirectly stabilizing the Hoogsteen conformation. A structure survey did not reveal Hoogsteen pairing near RNA apical loops making them unique to DNA. Our results demonstrate that Hoogsteen can be the dominant state even in naked unmodified duplex DNA and identify 5-CTT(T/C)AG-3 as a DNA-specific apical loop motif stabilized by Hoogsteen pairing. Hoogsteen base pairs may be prevalent in DNA hairpins forming during replication and transcription, with broad implications for the genomic landscape.

biochemistry↗

bioRxiv2024

Quantitative and systematic NMR measurements of sequence-dependent A-T Hoogsteen dynamics uncovers unique conformational specificity in the DNA double helix

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 R1{rho} 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.

biochemistry↗

bioRxiv2024

A specific phosphorylation-dependent conformational switch of SARS-CoV-2 nucleoprotein inhibits RNA binding

The nucleoprotein (N) of SARS-CoV-2 encapsidates the viral genome and is essential for viral function. The central disordered domain comprises a serine-arginine-rich domain (SR) that is hyperphosphorylated in infected cells. This modification is thought to regulate function of N, although mechanistic details remain unknown. We use time-resolved NMR to follow local and long-range structural changes occurring during hyperphosphorylation by the kinases SRPK1/GSK-3/CK1, thereby identifying a conformational switch that abolishes interaction with RNA. When 8 approximately uniformly-distributed sites are phosphorylated, the SR domain competitively binds the same interface as single-stranded RNA, resulting in RNA binding inhibition. Phosphorylation by PKA does not prevent RNA binding, indicating that the pattern resulting from the physiologically-relevant kinases is specific for inhibition. Long-range contacts between the RNA-binding, linker and dimerization domains are also abrogated, phenomena possibly related to genome packaging and unpackaging. This study provides insight into recruitment of specific host kinases to regulate viral function.

biochemistry↗

bioRxiv2023

NMR measurements of transient low-populated tautomeric and anionic Watson-Crick-like G·T/U in RNA:DNA hybrids: Implications for the fidelity of transcription and CRISPR/Cas9 gene editing

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 R1{rho} experiments, we show that dG*rU and dT*rG mismatches in two RNA:DNA hybrids transiently form tautomeric (Genol*T/U {rightleftarrows}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 ({Delta}pKa [~]0.4-0.9) 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.

biophysics↗