Search

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 k cat /K M 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., (© 2024. The Author(s).)

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

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., (© 2024. The Author(s).)

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Competing Interests: None declared.

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Objectives: The aim of this study was to describe the anatomy of the distal radioulnar ligament in the cat, using gross and histological sections from cadaveric feline carpi., Methods: Eight feline cadaveric distal radioulnar joints were included in the study, including six that were paraffin- and two that were polymethyl methacrylate-embedded. Each of the sections of the distal radioulnar joint and ligament were viewed macroscopically and microscopically using a dissection microscope and a standard light microscope with polarising capacity., Results: On gross examination, the distal radioulnar ligament could be seen as a triangular-shaped structure extending between the dorsal surface of the distal radius and ulna. The centre of the ligament had a greater density of tightly packed collagen fibres, while fibrocartilage was identified at the site of both the radial and ulnar entheses. Articular cartilage was noted to extend to the most proximal part of the bulbous portion of the distal ulna and corresponding axial aspect of the distal radius., Conclusions and Relevance: In the cat, there appears to be a less extensive interosseous component of the distal radioulnar ligament compared with the dog and cheetah. Instead, the ligament follows the articular surfaces of the distal radius and ulna. These anatomical differences may account for increased rotation of the feline antebrachium and have clinical implications, particularly with regard to the management of antebrachiocarpal joint injuries.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We describe the experimental methods and analysis to define the role of enzyme conformational changes in specificity based on published studies using DNA polymerases as an ideal model system. Rather than give details of how to perform transient-state and single-turnover kinetic experiments, we focus on the rationale of the experimental design and interpretation. We show how initial experiments to measure k cat and k cat /K m can accurately quantify specificity but do not define its underlying mechanistic basis. We describe methods to fluorescently label enzymes to monitor conformational changes and to correlate fluorescence signals with rapid-chemical-quench flow assays to define the steps in the pathway. Measurements of the rate of product release and of the kinetics of the reverse reaction complete the kinetic and thermodynamic description of the full reaction pathway. This analysis showed that the substrate-induced change in enzyme structure from an open to a closed state was much faster than rate-limiting chemical bond formation. However, because the reverse of the conformational change was much slower than chemistry, specificity is governed solely by the product of the binding constant for the initial weak substrate binding and the rate constant for the conformational change (k cat /K m =K 1 k 2 ) so that the specificity constant does not include k cat . The enzyme conformational change leads to a closed complex in which the substrate is bound tightly and is committed to the forward reaction. In contrast, an incorrect substrate is bound weakly, and the rate of chemistry is slow, so the mismatch is released from the enzyme rapidly. Thus, the substrate-induced-fit is the major determinant of specificity. The methods outlined here should be applicable to other enzyme systems., Competing Interests: Conflict of interest K.A.J. is president of KinTek Corporation, which provided the RQF-3 rapid quench flow and SF 300× Stopped-Flow instruments and KinTek Explorer software used in this study., (Copyright © 2023 Elsevier Inc. All rights reserved.)

A common mantra in drug discovery is that "You get what you screen for." This is not a promise that you will always get an effective drug candidate, but rather a warning that inaccuracies in your protocol for screening will more likely produce a compound that fails to be an effective candidate because it matches the properties of your screen, not the desired features of an ideal lead compound. It is with this in mind that we examine the current protocols for evaluating drug candidates and highlight some deficiencies while pointing the way to better methods. Many of the errors in data fitting can be rectified by abandoning the traditional equation-based data fitting methods and adopting the more rigorous mechanism-based fitting afforded by computer simulation based on numerical integration of rate equations. Using these methods bypasses the errors in judgement in choosing the appropriate equation for data fitting and the approximations required to derive those equations. In this chapter we outline the limitations and systematic errors in conventional methods of data fitting and illustrate the advantages of computer simulation and introduce the methods of analysis., (Copyright © 2023. Published by Elsevier Inc.)

The last review on transient-state kinetic methods in The Enzymes was published three decades ago (Johnson, K.A., 1992. The Enzymes, XX, 1-61). In that review the foundations were laid out for the logic behind the design and interpretation of experiments. In the intervening years the instrumentation has improved mainly in providing better sample economy and shorter dead times. More significantly, in 1992 we were just introducing methods for fitting data based on numerical integration of rate equations, but the software was slow and difficult to use. Today, advances in numerical integration methods for data fitting have led to fast and dynamic software, making it easy to fit data without simplifying approximations. This approach overcomes multiple disadvantages of traditional data fitting based on equations derived by analytical integration of rate equations, requiring simplifying approximations. Mechanism-based fitting using computer simulation resolves mechanisms by accounting for the concentration dependence of the rates and amplitudes of the reaction to find a set of intrinsic rate constants that reproduce the experimental data, including details about how the experiment was performed in modeling the data. Rather than discuss how to design and interpret rapid-quench and stopped-flow experiments individually, we now focus on how to fit them simultaneously so that the quench-flow data anchor the interpretation of fluorescence signals. Computer simulation streamlines the fitting of multiple experiments globally to yield a single unifying model to account for all available data., (Copyright © 2023. Published by Elsevier Inc.)

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 (10 9  M -1 s -1 , K d = 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., Competing Interests: Conflict of interest K. A. J. is the president of KinTek Corporation, which provided the RQF-3 rapid-quench flow and SF 300-X stopped-flow instruments and KinTek Explorer software used in this study. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus that causes COVID-19, continues to evolve resistance to vaccines and existing antiviral therapies at an alarming rate, increasing the need for new direct-acting antiviral drugs. Despite significant advances in our fundamental understanding of the kinetics and mechanism of viral RNA replication, there are still open questions regarding how the proofreading exonuclease (NSP10/NSP14 complex) contributes to replication fidelity and resistance to nucleoside analogs. Through single turnover kinetic analysis, we show that the preferred substrate for the exonuclease is double-stranded RNA without any mismatches. Double-stranded RNA containing a 3'-terminal remdesivir was hydrolyzed at a rate similar to a correctly base-paired cognate nucleotide. Surprisingly, single-stranded RNA or duplex RNA containing a 3'-terminal mismatch was hydrolyzed at rates 125- and 45-fold slower, respectively, compared to the correctly base-paired double-stranded RNA. These results define the substrate specificity and rate of removal of remdesivir for the exonuclease and outline rigorous kinetic assays that could help in finding next-generation exonuclease inhibitors or nucleoside analogs that are able to evade excision. These results also raise important questions about the role of the polymerase/exonuclease complex in proofreading during viral replication. Addressing these questions through rigorous kinetic analysis will facilitate the search for desperately needed antiviral drugs to combat COVID-19., Competing Interests: The authors declare the following competing financial interest(s): K.A.J. is president of KinTek Corporation, which provided the RQF-3 rapid quench flow instrument and KinTek Explorer software used in this study., (© 2022 The Authors. Published by American Chemical Society.)

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The current prognosis for successful return to function in koalas with appendicular fractures is poor despite being the most common fracture type to result in successful rehabilitation. The forelimb, particularly the humerus, plays a critical role in stabilisation and support while climbing trees. Successful rehabilitation therefore requires adequate internal stabilisation to promote bone healing and faster return to function. Current knowledge of koala limb bone morphometry is lacking and would provide useful clinical insight for future orthopaedic research, particularly with regards to recommendations regarding implant size and type. In this study microcomputed tomography (micro-CT) was used to describe bone length, internal and external diameters, and cortical thickness at five transverse levels along the humerus of skeletally mature koala cadavers. Qualitative descriptions were also made regarding bone features deemed clinically relevant to potential fracture repair techniques. Mean humeral length was 114.3 mm (95% CI 107.29-121.31 mm). Mediolateral diameters were greater than craniocaudal diameters at each measurement level, and the diaphysis has a distally tapering medullary cavity. Diaphyseal cortices were relatively homogenous with slight distal thickening, and medial cortices were thickest along the entire bone. The bone protuberances of the deltoid and supinator ridges projected most of the way down the lateral surface of the bone while the medial surface remained relatively uniform. Distal to the deltoid ridge the humerus curved caudally, terminating at a craniocaudally flattened distal epiphysis. Morphometric descriptions provided in this study will serve as a useful reference for future research, guiding orthopaedic surgery and improving prognosis of koala humeral fractures., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >10 9 variants led to no further improvements. Introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution., Competing Interests: Competing interests J.B., C.S.K., N.M., W.C.L., G.G., and E.S. are inventors on intellectual property related to this work, including the active patent US9975959B2 and the pending patents US20190350975A1 and US20210207110A1, which are assigned to the Board of Regents of the University of Texas System. G.G., E.S., X.M.Z., K.M., S.C., and M.M. have equity interest in Ikena Oncology, a company pursuing the commercial development of this technology. J.B., C.S.K., C.L., and E.S. have consulted for Ikena Oncology (previously Kyn Therapeutics). The remaining authors declare no competing interests.

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 pre–steady-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.

Objectives:  The aim of this study was to report the use of computed tomography (CT) for postoperative evaluation of the accuracy of sacroiliac reduction and minimally invasive screw fixation in a series of five cats., Methods:  Medical records between January 2016 and March 2017 of cats presenting to the author's institution were reviewed. Included were cats that had undergone minimally invasive sacroiliac screw fixation with a complete medical record and pre- and postoperative radiographs. Screw size was obtained from the medical records. CT images were acquired prospectively and evaluated to assess joint reduction, relative screw size and screw positioning., Results:  Six sacroiliac luxations and 6 screws were available. Fixation was achieved with either a 2.4 ( n  = 1) or 2.7 mm ( n  = 5), 316L stainless steel, cortical bone screw. Mean screw size as a proportion of sacral diameter was 47.7%. Sacroiliac reduction >90% in the craniocaudal plane and sacral screw purchase >60% of the sacral width were achieved in 3/5 cases. Mean dorsoventral screw angulation was 1.6 degrees (range: -9.7 to 11.7 degrees) and craniocaudal angulation was -4.5 degrees (range: -16.6 to 6.6 degrees). Complications included screw loosening in the one case of bilateral repair and penetration of the neural canal in one case which was not detected with postoperative radiographic evaluation., Clinical Significance:  CT evaluation provides a useful method for the assessment of sacroiliac reduction and the accuracy of screw placement., Competing Interests: None declared., (Thieme. All rights reserved.)

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 × 10 5 , 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., Competing Interests: Conflict of interest K. A. J. is president of KinTek Corporation, which provided the RQF-3 rapid quench flow instrument and KinTek Explorer software used in this study., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

CRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage 1-4 , and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood 5-7 . Although Cas9 variants with greater discrimination against mismatches have been designed 8-10 , these suffer from substantially reduced rates of on-target DNA cleavage 5,11 . 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., (© 2022. The Author(s).)

Objective:  The study aims to evaluate the biomechanical properties of feline femora with craniocaudal screw-hole defects of increasing diameter, subjected to three-point bending and torsion to failure at two different loading rates., Study Design:  Eighty femoral pairs were harvested from adult cat cadavers. For each bending and torsional experiment, there were five groups ( n  = 8 pairs) of increasing craniocaudal screw-hole defects (intact, 1.5 mm, 2.0 mm, 2.4 mm, 2.7mm). Mid-diaphyseal bicortical defects were created with an appropriate pilot drill-hole and tapped accordingly. Left and right femora of each pair were randomly assigned to a destructive loading protocol at low (10 mm/min; 0.5 degrees/s) or high rates (3,000 mm/min; 90 degrees/s) respectively. Stiffness, load/torque-to-failure, energy-to-failure and fracture morphology were recorded., Results:  Defect size to bone diameter ratio was significantly different between defect groups within bending and torsional experiments respectively (intact [0%; 0%], 1.5 mm [17.8%; 17.1%], 2.0 mm [22.8%; 23.5%], 2.4 mm [27.8%; 27.6%], 2.7 mm [31.1%; 32.4%]) ( p  < 0.001). No significant differences in stiffness and load/torque-to-failure were noted with increasing deficit sizes in all loading conditions. Screw-hole (2.7 mm) defects up to 33% bone diameter had a maximum of 20% reduction in bending and torsional strength compared with intact bone at both loading rates. Stiffness and load/torque-to-failure in both bending and torsion were increased in bones subjected to higher loading rates ( p  < 0.001)., Conclusion:  Screw-hole defects up to 2.7 mm did not significantly reduce feline bone failure properties in this ex vivo femoral study. These findings support current screw-size selection guidelines of up to 33% bone diameter as appropriate for use in feline fracture osteosynthesis., Competing Interests: None declared., (Thieme. All rights reserved.)

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., Competing Interests: Conflict of interest K. A. J. is president of KinTek Corporation, which provided the SF-300x stopped-flow instrument and KinTek Explorer software used in this study., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: None declared.

Competing Interests: None declared

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The RNA-dependent-RNA polymerase (RdRp) from SARS-CoV-2 is an important drug target because it is responsible for viral RNA genome replication. Efficient production of recombinant RdRp is important in screening antivirals to treat COVID-19. Here, we present our protocol for expression of tag-free replication complex proteins in E. coli and subsequent purification. Despite the added complexity of multiple purification steps, our methods provide greater activity, yield at lower cost, and are faster than baculovirus expression systems. For complete details on the use and execution of this protocol, please refer to Dangerfield et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Objective:  Cats rely on their forelimb mobility for everyday activities including climbing and grooming. Supination and pronation of the forelimb in cats are considered to primarily involve the antebrachium, rather than the carpus. Therefore, our null hypothesis was that there would be no movement of the carpal bones (radial carpal bone, ulnar carpal bone and accessory carpal bone) relative to the ulna during supination and pronation., Study Design:  Eight feline cadaveric forelimbs were rotated from supination to pronation in a jig and computed tomography was performed in the neutral, supinated and pronated positions. The individual carpal bones were segmented from computed tomography images of the supinated and pronated scans in each of the eight specimens. A feline ulna coordinate system was established and used to quantify the translations and rotations between bones of the proximal carpal row and antebrachium., Results:  After the carpus was rotated from the initial supinated position into pronation, there was significant translation (x, y and z axes) and rotation (x and y axes) of the proximal row of carpal bones based on absolute magnitude values. Given the differences in translations and rotations of the proximal row of carpal bones, our null hypothesis was rejected., Conclusion:  The proximal row of carpal bones translate and rotate independently from the ulna in the cat during pronation of the antebrachium. This may have future implications in the diagnosis and management of feline carpal injuries involving the antebrachiocarpal joint., Competing Interests: None declared., (Thieme. All rights reserved.)

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.

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 Mg 2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg 2+ 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 Mg 2+ concentration. Subsequently, the second Mg 2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (K d  = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg 2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg 2+ concentrations. An increase of the Mg 2+ concentration from 0.25 to 10 mM increases nucleotide specificity (k cat /K m ) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg 2+ binds very weakly (K d ≤ 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 Mg 2+ . 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., Competing Interests: Conflict of interest K. A. J. is the President of KinTek Corp, which provided the AutoSF-120 stopped-flow, RQF-3 rapid-quench-flow, and KinTek Explorer software used in this study., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Conflict of interest K. A. J. is president of KinTek Corporation, which provided the SF300x stopped-flow and RQF-3 rapid-quench-flow instruments, and KinTek Explorer software used in this study., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Nucleotide analogs are the cornerstone of direct acting antivirals used to control infection by RNA viruses. Here we review what is known about existing nucleotide/nucleoside analogs and the kinetics and mechanisms of RNA and DNA replication, with emphasis on the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) in comparison to HIV reverse transcriptase and Hepatitis C RdRp. We demonstrate how accurate kinetic analysis reveals surprising results to explain the effectiveness of antiviral nucleoside analogs providing guidelines for the design of new inhibitors., (Copyright © 2021 Elsevier Inc. All rights reserved.)