Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.

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.

History of advances in enzyme kinetic methods: From minutes to milliseconds.

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.

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

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.

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.

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

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 kcat and kcat/Km 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 (kcat/Km=K1k2) so that the specificity constant does not include kcat. 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.

Anatomy of the distal radioulnar ligament in cats.

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.

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.

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.

Substrate Specificity and Kinetics of RNA Hydrolysis by SARS-CoV-2 NSP10/14 Exonuclease.

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.

Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.

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 >109 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.