DNA targeting by compact Cas9d and its resurrected ancestor.

Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion.

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

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.

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.

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.

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.

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.

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 k and k/K can accurately quantify specificity but do not define its underlying mechanistic basis.

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.

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.

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 >10 variants led to no further improvements.

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.