5mga
From Proteopedia
Structure of the Cpf1 endonuclease R-loop complex after DNA cleavage
Structural highlights
Function[CPF1_FRATN] CRISPR (clustered regularly interspaced short palindromic repeat), is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). This protein is a single-RNA guided endonuclease that is also capable of guiding crRNA processing; correct processing of pre-crRNA requires only this protein and the CRISPR locus (PubMed:26422227). When this protein is expressed in E.coli it prevents plasmids homologous to the first CRISPR spacer from transforming, showing it is responsible for plasmid immunity (PubMed:26422227). Has dsDNA endonuclease activity, results in staggered 5-base 5' overhangs 18 and 23 bases downstream of the PAM (protospacer adjacent motif) on the non-targeted and targeted strands respectively (PubMed:26422227).[1] Publication Abstract from PubMedCpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA-DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic conformation, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a 'rail' shape and 'flap-on' conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the 'septum', separating the displaced PAM strand and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage.,Stella S, Alcon P, Montoya G Nature. 2017 Jun 22;546(7659):559-563. doi: 10.1038/nature22398. Epub 2017 May, 31. PMID:28562584[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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Categories: Montoya, G | Stella, S | Cpf1 | Crispr | Fncpf1 | Hydrolase | R-loop
