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) signalling molecules, potentiating a powerful immune response by activating downstream effector proteins, leading to viral clearance, cell dormancy or death. Here we describe the structure and mechanism of a cOA-activated CRISPR defence DNA endonuclease, CRISPR ancillary nuclease 1 (Can1). Can1 has a unique monomeric structure with two CRISPR associated Rossman fold (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 (cA4) molecule bound at the core of the protein. cA4 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.
Highlights
The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes
As we lacked a crystal structure of apo Can[1], we investigated the solution state of apo Can[1] and Can[1] bound to cA4 using small angle X-ray scattering (SAXS)
The structure of the Can[1] enzyme clearly demonstrates that the two halves of the protein, comprising a CRISPR associated Rossman fold (CARF) domain N-terminal to a nuclease fold domain, are related to one another
Summary
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) signalling molecules, potentiating a powerful immune response by activating downstream effector proteins, leading to viral clearance, cell dormancy or death. Can[1] has a unique monomeric structure with two CRISPR associated Rossman fold (CARF) domains and two DNA nuclease-like domains. Type III (Csm/Cmr) CRISPR systems utilise a cyclase domain in the Cas[10] subunit to generate cyclic oligoadenylate (cOA) by polymerising ATP1–3. COA is a potent antiviral second messenger that sculpts the anti-viral response by binding to and activating CRISPR associated Rossman fold (CARF) family proteins[5]. The T. thermophilus Cmr (type III-B) complex was one of the first studied biochemically, revealing the distinctive cleavage of target RNA with 6 nucleotide spacing[18,19].
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