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Seminar: Guillermo Montoya: Crystal structure of the Cpf1 R-loop complex after target DNA cleavage

27.04.2017 | Lisbeth Heilesen

Dato fre 28 apr
Tid 15:00 16:00
Sted Science Park, Conference Room, 3130-303

Guillermo Montoya heads the upcoming Titan-Krios cryo-EM facility at the University of Copenhagen and presents a talk on his current work on genome/DNA modifying enzymes using also X-ray crystallography.

Crystal structure of the  Cpf1 R-loop complex after target DNA cleavage

Prof. Guillermo Montoya

University of Copenhagen, the Novo Nordisk Foundation Center for Protein Research

Cpf1 is a single RNA-guided endonuclease of class 2 type V CRISPR-Cas system, emerging as a powerful genome editing tool 1,2. To provide insight into its DNA targeting mechanism, we have determined the crystal structure of Francisella novicida Cpf1 (FnCpf1) in complex with the triple strand R-loop formed after target DNA cleavage. The structure reveals a unique machinery for target DNA unwinding to form a crRNA-DNA hybrid and a displaced DNA strand inside FnCpf1. The protospacer adjacent motif (PAM) is recognised by the PAM interacting (PI) domain. In this domain, the conserved K667, K671 and K677 are arranged in a dentate manner in a loop-lysine helix-loop motif (LKL). The helix is inserted at a 45º angle to the dsDNA longitudinal axis. Unzipping of the dsDNA in a cleft arranged by acidic and hydrophobic residues facilitates the hybridization of the target DNA strand with crRNA. K667 initiates unwinding by pushing away the guanine after the PAM sequence of the dsDNA. The PAM ssDNA is funnelled towards the nuclease site, which is located 70 Å away, through a hydrophobic protein cavity with basic patches that interact with the phosphate backbone. In this catalytically active conformation the PI and the helix-loop-helix (HLH) motif in the REC1 domain adopt a “rail shape” and “flap-on” conformations, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms a “septum” that separates the displaced PAM strand and the crRNA-DNA hybrid, avoiding re-annealing of the DNA. Mutations in key residues reveal a novel mechanism to determine the DNA product length, thereby linking the PAM and DNA nuclease sites. Our study reveals a singular working model of RNA-guided DNA cleavage by Cpf1, opening up new avenues for engineering this genome modification system.

Time: Friday April 28th at 15.00

Place: Dept. Molecular Biology and Genetics, Gustav Wieds Vej 10C, Conference Room (3130-303)

There will be refreshments during and after the talk


Prof. Poul Nissen, PhD

Aarhus University
Dept. Molecular Biology and Genetics Gustav Wieds Vej 10C, DK - 8000 Aarhus C, Denmark
Tel. +45 2899 2295 http://pure.au.dk/portal/da/pn@mbg.au.dk