Remdesivir is a delayed translocation inhibitor of SARS-CoV-2 replication.

Remdesivir is a nucleoside analog approved by the US FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryo-EM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of 3 copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of 3 bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

Structural basis for mismatch surveillance by CRISPR-Cas9.

CRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1-4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5-7. Although Cas9 variants with greater discrimination against mismatches have been designed8-10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA-DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA-DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.

Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting.

Loop-mediated isothermal amplification (LAMP) has proven to be easier to implement than PCR for point-of-care diagnostic tests. However, the underlying mechanism of LAMP is complicated and the kinetics of the major steps in LAMP have not been fully elucidated, which prevents rational improvements in assay development. Here we present our work to characterize the kinetics of the elementary steps in LAMP and show that: (i) strand invasion / initiation is the rate-limiting step in the LAMP reaction; (ii) the loop primer plays an important role in accelerating the rate of initiation and does not function solely during the exponential amplification phase and (iii) strand displacement synthesis by Bst-LF polymerase is relatively fast (125 nt/s) and processive on both linear and hairpin templates, although with some interruptions on high GC content templates. Building on these data, we were able to develop a kinetic model that relates the individual kinetic experiments to the bulk LAMP reaction. The assays developed here provide important insights into the mechanism of LAMP, and the overall model should be crucial in engineering more sensitive and faster LAMP reactions. The kinetic methods we employ should likely prove useful with other isothermal DNA amplification methods.

High fidelity DNA strand-separation is the major specificity determinant in DNA methyltransferase CcrM's catalytic mechanism.

Strand-separation is emerging as a novel DNA recognition mechanism but the underlying mechanisms and quantitative contribution of strand-separation to fidelity remain obscure. The bacterial DNA adenine methyltransferase, CcrM, recognizes 5'GANTC'3 sequences through a DNA strand-separation mechanism with unusually high selectivity. To explore this novel recognition mechanism, we incorporated Pyrrolo-dC into cognate and noncognate DNA to monitor the kinetics of strand-separation and used tryptophan fluorescence to follow protein conformational changes. Both signals are biphasic and global fitting showed that the faster phase of DNA strand-separation was coincident with the protein conformational transition. Non-cognate sequences did not display strand-separation and methylation was reduced > 300-fold, providing evidence that strand-separation is a major determinant of selectivity. Analysis of an R350A mutant showed that the enzyme conformational step can occur without strand-separation, so the two events are uncoupled. A stabilizing role for the methyl-donor (SAM) is proposed; the cofactor interacts with a critical loop which is inserted between the DNA strands, thereby stabilizing the strand-separated conformation. The results presented here are broadly applicable to the study of other N6-adenine methyltransferases that contain the structural features implicated in strand-separation, which are found widely dispersed across many bacterial phyla, including human and animal pathogens, and some Eukaryotes.

Leveraging intrinsic flexibility to engineer enhanced enzyme catalytic activity.

Dynamic motions of enzymes occurring on a broad range of timescales play a pivotal role in all steps of the reaction pathway, including substrate binding, catalysis, and product release. However, it is unknown whether structural information related to conformational flexibility can be exploited for the directed evolution of enzymes with higher catalytic activity. Here, we show that mutagenesis of residues exclusively located at flexible regions distal to the active site of Homo sapiens kynureninase (HsKYNase) resulted in the isolation of a variant (BF-HsKYNase) in which the rate of the chemical step toward kynurenine was increased by 45-fold. Mechanistic pre­steady-state kinetic analysis of the wild type and the evolved enzyme shed light on the underlying effects of distal mutations (>10 Å from the active site) on the rate-limiting step of the catalytic cycle. Hydrogen-deuterium exchange coupled to mass spectrometry and molecular dynamics simulations revealed that the amino acid substitutions in BF-HsKYNase allosterically affect the flexibility of the pyridoxal-5'-phosphate (PLP) binding pocket, thereby impacting the rate of chemistry, presumably by altering the conformational ensemble and sampling states more favorable to the catalyzed reaction.

Kinetics of DNA strand transfer between polymerase and proofreading exonuclease active sites regulates error correction during high-fidelity replication.

We show that T7 DNA polymerase (pol) and exonuclease (exo) domains contribute to selective error correction during DNA replication by regulating bidirectional strand transfer between the two active sites. To explore the kinetic basis for selective removal of mismatches, we used a fluorescent cytosine analog (1,3-diaza-2-oxophenoxazine) to monitor the kinetics of DNA transfer between the exo and pol sites. We globally fit stopped-flow fluorescence and base excision kinetic data and compared results obtained with ssDNA versus duplex DNA to resolve how DNA transfer governs exo specificity. We performed parallel studies using hydrolysis-resistant phosphorothioate oligonucleotides to monitor DNA transfer to the exo site without hydrolysis. ssDNA binds to the exo site at the diffusion limit (109 M-1 s-1, Kd = 40 nM) followed by fast hydrolysis of the 3'-terminal nucleotide (>5000 s-1). Analysis using duplex DNA with a 3'-terminal mismatch or a buried mismatch exposed a unique intermediate state between pol and exo active sites and revealed that transfer via the intermediate to the exo site is stimulated by free nucleoside triphosphates. Transfer from the exo site back to the pol site after cleavage is fast and efficient. We propose a model to explain why buried mismatches are removed faster than single 3'-terminal mismatches and thereby provide an additional opportunity for error correction. Our data provide the first comprehensive model to explain how DNA transfer from pol to exo active sites and back again after base excision allow efficient selective mismatch removal during DNA replication to improve fidelity by more than 1000-fold.

Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase.

Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 105, fidelity is further enhanced several orders of magnitude by a 3'-5' proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations.

Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid.

High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension with the correct nucleotide, the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. The simulations suggest that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, nonideal base pairing, and a large increase in the distance from the 3'-OH group of the primer strand to the α-phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high-fidelity DNA polymerases.

Structure of the DNA repair helicase XPD.

The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalyzing DNA duplex opening localized to the transcription start site or site of DNA damage, respectively. XPD has a 5' to 3' polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigmentosum, trichothiodystrophy, and Cockayne's syndrome, characterized by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 A crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans.

Pyrophosphate release acts as a kinetic checkpoint during high-fidelity DNA replication by the Staphylococcus aureus replicative polymerase PolC.

Bacterial replication is a fast and accurate process, with the bulk of genome duplication being catalyzed by the α subunit of DNA polymerase III within the bacterial replisome. Structural and biochemical studies have elucidated the overall properties of these polymerases, including how they interact with other components of the replisome, but have only begun to define the enzymatic mechanism of nucleotide incorporation. Using transient-state methods, we have determined the kinetic mechanism of accurate replication by PolC, the replicative polymerase from the Gram-positive pathogen Staphylococcus aureus. Remarkably, PolC can recognize the presence of the next correct nucleotide prior to completing the addition of the current nucleotide. By modulating the rate of pyrophosphate byproduct release, PolC can tune the speed of DNA synthesis in response to the concentration of the next incoming nucleotide. The kinetic mechanism described here would allow PolC to perform high fidelity replication in response to diverse cellular environments.

Conformational dynamics during high-fidelity DNA replication and translocation defined using a DNA polymerase with a fluorescent artificial amino acid.

We address the role of enzyme conformational dynamics in specificity for a high-fidelity DNA polymerase responsible for genome replication. We present the complete characterization of the conformational dynamics during the correct nucleotide incorporation forward and reverse reactions using stopped-flow and rapid-quench methods with a T7 DNA polymerase variant containing a fluorescent unnatural amino acid, (7-hydroxy-4-coumarin-yl) ethylglycine, which provides a signal for enzyme conformational changes. We show that the forward conformational change (>6000 s-1) is much faster than chemistry (300 s-1) while the enzyme opening to allow release of bound nucleotide (1.7 s-1) is much slower than chemistry. These parameters show that the conformational change selects a correct nucleotide for incorporation through an induced-fit mechanism. We also measured conformational changes occurring after chemistry and during pyrophosphorolysis, providing new insights into processive polymerization. Pyrophosphorolysis occurs via a conformational selection mechanism as the pyrophosphate binds to a rare pretranslocation state of the enzyme-DNA complex. Global data fitting was achieved by including experiments in the forward and reverse directions to correlate conformational changes with chemical reaction steps. This analysis provided well-constrained values for nine rate constants to establish a complete free-energy profile including the rates of DNA translocation during processive synthesis. Translocation does not follow Brownian ratchet or power stroke models invoking nucleotide binding as the driving force. Rather, translocation is rapid and thermodynamically favorable after enzyme opening and pyrophosphate release, and it appears to limit the rate of processive synthesis at 4 °C.

Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis.

Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase. We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. An increase of the Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV reverse transcriptase binds only two metal ions prior to incorporation of a correct base pair. Molecular dynamics simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. Molecular dynamics simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions in the specificity of DNA polymerases.

The effect of CT and MRI with and without arthrography on the appearance of the feline carpal ligaments.

BACKGROUND: The current diagnosis of feline carpal injuries is based on radiographic examination including stress views and computed tomography; however, these techniques do not allow for direct evaluation of the carpal ligaments. The purpose of this cadaveric study was to assess the ability of CT arthrography (CTA) and MR arthrography (MRA) to provide this information using a single contrast mixture. A protocol for intra-articular injection of the feline carpus was also described. A contrast solution containing gadolinium and iohexol with a 50% gadolinium solution (Magnevist-gadolinium 0.5 mmol/mL diluted to a 0.05 mmol/mL solution) and 50% of iodine (Iohexol-iodine 300mgI/mL) was injected into the antebrachiocarpal and middle carpal joints of feline carpi using fluoroscopic guidance. RESULTS: CTA allowed for identification of intra-articular ligaments and the silhouette of select extra-articular ligaments when there was adequate joint distension, however it was not considered to be superior to MRI. MRA allowed for improved identification of the dorsal radiocarpal, accessorioulnocarpal, accessorioquartile, short ulnar and short radial collateral ligaments. CONCLUSION: In this ex-vivo study, combined CTA and MRA enhanced the appearance of the feline carpal ligaments and may provide a foundation for future studies in the diagnosis of carpal injuries.

Rate-limiting pyrophosphate release by hepatitis C virus polymerase NS5B improves fidelity.

The hepatitis C virus RNA-dependent RNA polymerase NS5B is responsible for the replication of the viral genome. Previous studies have uncovered NTP-mediated excision mechanisms that may be responsible for aiding in maintaining fidelity (the frequency of incorrect incorporation events relative to correct), but little is known about the fidelity of NS5B. In this study, we used transient-state kinetics to examine the mechanistic basis for polymerase fidelity. We observe a wide range of efficiency for incorporation of various mismatched base pairs and have uncovered a mechanism in which the rate constant for pyrophosphate release is slowed for certain misincorporation events. This results in an increase in fidelity against these specific misincorporations. Furthermore, we discover that some mismatches are highly unfavorable and cannot be observed under the conditions used here. The calculated fidelity of NS5B ranges between 10-4-10-9 for different mismatches.

Optimized incorporation of an unnatural fluorescent amino acid affords measurement of conformational dynamics governing high-fidelity DNA replication.

DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication.

Resistance to excision determines efficiency of hepatitis C virus RNA-dependent RNA polymerase inhibition by nucleotide analogs.

NS5B is the RNA-dependent RNA polymerase that catalyzes the replication of the hepatitis C virus genome. It is a major target for antiviral drugs including nucleoside analogs, such as the prodrugs mericitabine and sofosbuvir, which get metabolized to 2'-fluoro-2'C-methylcytidine-5'-triphosphate and 2'-fluoro-2'C-methyluridine-5'-triphosphate, respectively. These analogs act as chain terminators after they are incorporated during RNA synthesis. Recently, it has been shown that NS5B can efficiently remove chain terminators by a nucleotide-mediated excision reaction that rescues RNA synthesis. In this study, we use transient-state kinetics to understand the efficiency of inhibition for five nucleoside analogs. We show that CTP analogs are readily incorporated into a growing primer by NS5B but are also efficiently excised. In contrast, although UMP analogs are more slowly incorporated, the excision of UMP is slow and inefficient, and modifications to the 2'-carbon of the UTP ribose ring further decreased rates of excision to an undetectable level. Taken together, these data suggest that the clinical effectiveness of sofosbuvir is largely a function of being intractable to nucleotide-mediated excision compared with similar nucleoside analogs.

High throughput quantification of short nucleic acid samples by capillary electrophoresis with automated data processing.

Analysis of catalytic activity of nucleic acid enzymes is crucial for many applications, ranging from biotechnology to the search for antiviral drugs. Commonly used analytical methods for quantifying DNA and RNA reaction products based on slab-gel electrophoresis are limited in throughput, speed, and accuracy. Here we report the optimization of high throughput methods to separate and quantify short nucleic acid reaction products using DNA sequencing instruments based on capillary electrophoresis with fluorescence detection. These methods afford single base resolution without requiring extensive sample preparation. Additionally, we show that the utility of our system extends to quantifying RNA products. The efficiency and reliability of modern instruments offers a large increase in throughput but complications due to variations in migration times between capillaries required us to develop a computer program to normalize the data and quantify the products for automated kinetic analysis. The methods presented here greatly increase sample throughput and accuracy and should be applicable to many nucleic acid enzymes.

The effect of plate fixation on supination and pronation of the feline antebrachium: a model of pancarpal arthrodesis.

BACKGROUND: Pancarpal arthrodesis is purported to limit supination and pronation of the feline antebrachium. The objective of this study was to investigate whether plate fixation of the radius to the carpus and metacarpus limits supination and pronation of the ulna relative to the radius as a model for pancarpal arthrodesis in the cat. Eight feline cadaveric forelimbs were rotated from supination to pronation in a testing jig and CT (computed tomography) was performed in the neutral, supinated and pronated positions. A locking plate was then secured dorsally to the radius, radial carpal bone and metacarpal III of each of the limbs. CT was repeated in each of the testing positions following plate application. The radius and ulna of the control specimens, and the radius, ulna and plate of the plated specimens were then segmented using software. Alignment of the bones to the radius in the control specimens, and to the plate in the plated specimens was used to compare the changes in degrees of movement of the ulna relative to the radius in dorsal, sagittal and transverse planes. RESULTS: Based on the results of the paired t test, there was no significant difference in degrees of movement, or total range of motion between control and plated specimens in supinated and pronated testing conditions. CONCLUSION: The results of this ex-vivo study indicate that under the testing conditions employed, plate fixation of the radius to the carpus and metacarpus does not limit supination and pronation of the feline antebrachium.

Visible Light Mediated Bidirectional Control over Carbonic Anhydrase Activity in Cells and in Vivo Using Azobenzenesulfonamides.

Two azobenzenesulfonamide molecules with thermally stable cis configurations resulting from fluorination of positions ortho to the azo group are reported that can differentially regulate the activity of carbonic anhydrase in the trans and cis configurations. These fluorinated probes each use two distinct visible wavelengths (520 and 410 or 460 nm) for isomerization with high photoconversion efficiency. Correspondingly, the cis isomer of these systems is highly stable and persistent (as evidenced by structural studies in solid and solution state), permitting regulation of metalloenzyme activity without continuous irradiation. Herein, we use these probes to demonstrate the visible light mediated bidirectional control over the activity of zinc-dependent carbonic anhydrase in solution as an isolated protein, in intact live cells and in vivo in zebrafish during embryo development.