Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2

To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2–crRNA–DNA complexes and a Cas12i2–crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1–8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9–15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.

Alkali metal ion catalysis and inhibition in alkaline ethanolysis of O-Y-substituted-phenyl O-phenyl thionocarbonates: contrasting M+ ion effects upon changing electrophilic centre from C=O to C=S

Pseudo-first-order rate constants (kobsd) were measured for nucleophilic substitution reactions of O-Y-substituted-phenyl O-phenyl thionocarbonates (4a–4h) with alkali metal ethoxides (EtOM, M = Li, Na, or K) in anhydrous ethanol at 25.0 ± 0.1 °C. Plots of kobsd vs. [EtOM] exhibited upward curvature for the reaction of O-4-nitrophenyl O-phenyl thionocarbonate (4a) with EtOK in the presence of 18-crown-6-ether (18C6), but showed downward curvature for the reaction with EtOLi, indicating that the reaction is catalyzed by the 18C6-crowned K+ ion, but is inhibited by Li+ ion. The kobsd values were dissected into kEtO− and kEtOM, the second-order rate constant for the reaction with dissociated EtO− and ion-paired EtOM, respectively. The reactivity of EtOM toward 4a increases in the order EtOLi < EtONa < EtO− < EtOK < EtOK/18C6, which is in contrast to that reported previously for the corresponding reaction of 4-nitrophenyl phenyl carbonate (a C=O analogue of 4a), e.g., EtO− ≈ EtOK/18C6 < EtOLi < EtONa < EtOK. The reaction mechanism, including the transition-state model and the origin of the contrasting reactivity patterns found for the reactions of the C=O and C=S compounds, are discussed.