Abstract Cells and organisms have a wide range of mechanisms to defend against infection by viruses and other mobile genetic elements (MGE). Type III CRISPR systems detect foreign RNA and typically generate cyclic oligoadenylate (cOA) second messengers that bind to ancillary proteins with CARF (CRISPR associated Rossman fold) domains. This results in the activation of fused effector domains for antiviral defence. The best characterised CARF family effectors are the Csm6/Csx1 ribonucleases and DNA nickase Can1. Here we investigate a widely distributed CARF family effector with a nuclease domain, which we name Can2 (CRISPR ancillary nuclease 2). Can2 is activated by cyclic tetra-adenylate (cA4) and displays both DNase and RNase activity, providing effective immunity against plasmid transformation and bacteriophage infection in Escherichia coli. The structure of Can2 in complex with cA4 suggests a mechanism for the cA4-mediated activation of the enzyme, whereby an active site cleft is exposed on binding the activator. These findings extend our understanding of type III CRISPR cOA signalling and effector function.
Abstract The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes. On binding invading RNA species, Type III CRISPR systems generate cyclic oligoadenylate (cOA) molecules which act as a second messenger, signalling infection and potentiating a powerful immune response by activating a range of downstream effector proteins that can lead to viral clearance, cell dormancy or death. Only one type of effector enzyme has been studied – the Csm6/Csx1 ribonuclease domain, and the mechanism of cOA activation is not understood at a molecular level. Here we describe the structure and mechanism of a novel cOA-activated CRISPR defence DNA endonuclease, Can1 (“CRISPR ancillary nuclease 1”). Can1 has a unique monomeric structure with two CRISPR associated Rossman fold (CARF) CARF domains and two DNA nuclease-like domains. The crystal structure of the enzyme has been captured in the activated state, with a cyclic tetra-adenylate (cA 4 ) molecule bound at the core of the protein. cA 4 binding reorganises the structure to license a metal-dependent DNA nuclease activity specific for nicking of supercoiled DNA. DNA nicking by Can1 is predicted to slow down viral replication kinetics by leading to the collapse of DNA replication forks.
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