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Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9.

ABSTRACT: The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures of precatalytic, postcatalytic and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg²⁺. In the precatalytic state, Cas9 adopts the 'checkpoint' conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the postcatalytic state involves a dramatic ~34-Å swing of the HNH domain and disorder of the REC2 recognition domain. The postcatalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the precatalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and the RNA-DNA hybrid provide new insights into the mechanism of genome editing by Cas9.

SUBMITTER: Zhu X

PROVIDER: S-EPMC6842131 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism.

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| S-EPMC7196072 | biostudies-literature

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Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase.

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Author Correction: Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism.

Project description:An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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| S-EPMC4752722 | biostudies-literature

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