Slow PPi release enhances fidelity of the SARS-CoV-2 RNA dependent RNA polymerase

Journal of Biological Chemistry · 2026-04-17

<h2>Abstract</h2> Viral RNA-dependent RNA polymerases (RdRps) must balance replication speed with fidelity, preserving genome integrity while permitting enough variability for viral adaptation. The SARS-CoV-2 RdRp complex (NSP12/7/8), achieves this through the interplay of its intrinsic replication fidelity and a potential proofreading exonuclease complex (NSP10/14). Here, we comprehensively quantify the intrinsic fidelity of the SARS-CoV-2 RdRp through direct pre-steady-state kinetic analyses of nucleotide incorporation across all possible templating bases paired with incoming nucleotides. We discovered substantial variation in discrimination against mismatches ranging from one error in 10³ to 10⁸ (median of 10⁵). Crucially, our data reveal a slow pyrophosphate release step that significantly enhances fidelity by effectively introducing a kinetic checkpoint after nucleotide incorporation. The error rates we measured for the RdRp align closely with observed <i>in vivo</i> mutation rates, suggesting that the exonuclease complex may play a less critical role than previously assumed in correcting mistakes during polymerization. These insights advance our understanding of SARS-CoV-2 replication fidelity, and the role of various subcomplexes in genome maintenance and adaptation.

Kinetic mechanism of Renilla luciferase guides induced-fit engineering for improved bioluminescence

bioRxiv (Cold Spring Harbor Laboratory) · 2025-09-18 · 1 citations

Renilla luciferase (RLuc) remains one of the most popular bioluminescence reporters, but its molecular principle has yet to be fully understood. Here, we reveal a detailed kinetic mechanism of the RLuc catalytic cycle which uncovers multiple limiting factors: (i) an oxygen-induced irreversible inactivation, (ii) a low oxygen saturation, and (iii) rate-limiting induced-fit conformational dynamics coupled with the product release. Furthermore, we could determine the actual enzyme k cat value at all saturating substrates to be 22 s -1 . This value is 5-fold higher than the previously reported apparent k cat values determined at physiological, non-saturating oxygen concentration. Our integrative analysis by transient kinetics, X-ray crystallography, and molecular dynamics linked the rate-limiting flexible enzyme opening to the dynamics of the loops surrounding the active site, which prompted targeted engineering of this limiting step by loop grafting. The resulting variant AncFT-L14 (AncFT7) showed a prolonged stable light emission thanks to the selectively improved induced-fit kinetics. Additional characterization of AncFT-L14 identified increased catalytic efficiency k cat / K m , product inhibition factor K p / K m , and a glow-type signal characteristic. Our results provide mechanistic details of RLuc catalysis and will govern future enzyme engineering to design the next generations of bioluminescence-based tools.

Mechanistic conformational and substrate selectivity profiles emerging in the evolution of enzymes via parallel trajectories

Nature Communications · 2024-08-16 · 9 citations

Laboratory evolution studies have demonstrated that parallel evolutionary trajectories can lead to genetically distinct enzymes with high activity towards a non-preferred substrate. However, it is unknown whether such enzymes have convergent conformational dynamics and mechanistic features. To address this question, we use as a model the wild-type Homo sapiens kynureninase (HsKYNase), which is of great interest for cancer immunotherapy. Earlier, we isolated HsKYNase_66 through an unusual evolutionary trajectory, having a 410-fold increase in the kcat/KM for kynurenine (KYN) and reverse substrate selectivity relative to HsKYNase. Here, by following a different evolutionary trajectory we generate a genetically distinct variant, HsKYNase_93D9, that exhibits KYN catalytic activity comparable to that of HsKYNase_66, but instead it is a “generalist” that accepts 3’-hydroxykynurenine (OH-KYN) with the same proficiency. Pre-steady-state kinetic analysis reveals that while the evolution of HsKYNase_66 is accompanied by a change in the rate-determining step of the reactions, HsKYNase_93D9 retains the same catalytic mechanism as HsKYNase. HDX-MS shows that the conformational dynamics of the two enzymes are markedly different and distinct from ortholog prokaryotic enzymes with high KYN activity. Our work provides a mechanistic framework for understanding the relationship between evolutionary mechanisms and phenotypic traits of evolved generalist and specialist enzyme species. In this work, the authors show that two parallel and distinct evolutionary pathways can lead to genetically diverse enzyme species that harbour similar catalytic activities against the desired substrate, modulated by distinct conformational dynamics during catalysis.

Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9

Nature Communications · 2024-04-30 · 55 citations

CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.

High-Strain-Rate ~ 106/s Response of 304 Stainless Steel at Various Strains

2024-10-23

The effect of high strain rate at controlled strain levels has been investigated on 304 stainless steel. This study implements a radial shock loading design with a reliable specimen recovery. Strains were measured by plating circle grids on a split anvil design and measured after shock loading. The strain levels were controlled by varying the momentum trap geometries. The shock wave profile impinging and traveling through the specimen as obtained by hydrocode calculations, is in fact a shear wave. This shock loading design yields a specimen with a gradient of shock levels up to 1.7 Mbars at a pulse duration of less than one microsecond. The pressure range is achieved nominally independent of the strain level. With this strain pressure independence we were able to study the amount of strain induced α’-martensite generally associated with 304 stainless steel deformation experiments.

Abstract 1968 Dynamics of Exonuclease Proofreading by T7 DNA Polymerase During DNA Replication

Journal of Biological Chemistry · 2024-03-01

In this study we explore the interplay of polymerase and exonuclease active sites of T7 DNA polymerase in selective error correction during high fidelity DNA replication. First, we characterize the substrate specificity of the proofreading exonuclease domain in a high-fidelity DNA polymerase, showing efficient proofreading of terminal mismatches and surprisingly, even greater efficiency for mismatches buried by correct bases suggesting that the polymerase may correct some mistakes by first extending them with the correct base. We then developed a homology model for the DNA primer strand in the exonuclease active site showing that the DNA must backtrack and 3 bases melt for the primer to partition to the exonuclease active site for hydrolysis. Based on these results we designed a DNA substrate containing the fluorescent cytosine analog tCo at the n-2 position to measure kinetics of proofreading on physiologically relevant substrates. Combining pre-steady state stopped flow fluorescence measurements of strand separation with single turnover rapid quench experiments allowed us to obtain a global fit to elucidate the rate constants and kinetic mechanism for high fidelity error correction. These studies revealed a unique intermediate state and stimulation of transfer to the exo site by nucleoside triphosphates. Hydrolysis-resistant oligonucleotides track DNA transfer without hydrolysis. Our proposed model explains faster removal of buried mismatches than single 3′-terminal mismatches, providing an additional avenue for error correction. This comprehensive model demonstrates how DNA transfer, coupled with base excision, facilitates efficient selective mismatch removal during DNA replication, enhancing fidelity by over 1000-fold.

Single-turnover kinetic analysis of non-LTR retrotransposition defines the mechanism and rate constants governing each step

Research Square · 2024-09-04

You get what you screen for: Standards for experimental design and data fitting in drug discovery

Methods in enzymology on CD-ROM/Methods in enzymology · 2023-01-01

Unraveling the mechanisms of PAMless DNA interrogation by SpRY Cas9

bioRxiv (Cold Spring Harbor Laboratory) · 2023-06-22 · 9 citations

Abstract CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an “NGG” protospacer-adjacent motif (PAM) sequence immediately adjacent to the DNA target limits the number of editable genes. To overcome the PAM requirement, a recently developed Cas9 variant, called SpRY-Cas9 was engineered to be “PAMless” (1, 2). However, the molecular mechanisms of how SpRY can recognize all potential PAM sequences and still accurately identify DNA targets have not been investigated. Here, we combined enzyme kinetics, cryo-EM, and single-molecule imaging to determine how SpRY interrogates DNA and recognizes target sites for cleavage. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region of SpRY, which facilitates tight binding to off-target DNA sequences. Once SpRY correctly identifies a target site, nuclease activation occurs ∼1,000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Insights gained from our intermediate structures prompted rationally designed mutants with improved DNA cleavage efficiency. Our findings shed light on the molecular mechanisms of PAMless genome editing with SpRY and provide a framework for the design of future genome editing tools with improved versatility, precision, and efficiency.

Design and interpretation of experiments to establish enzyme pathway and define the role of conformational changes in enzyme specificity

Methods in enzymology on CD-ROM/Methods in enzymology · 2023-01-01 · 2 citations