SNAP output for PDB entry 5xvo [SNAP web server]

PDB-id

5xvo; SNAP-derived features in text and JSON formats; DNAproDB

Class

immune system

Method

X-ray (3.1 Å)

Summary

E. fae cas1-cas2-prespacer-target ternary complex revealing DNA sampling and half-integration states

Reference

Xiao Y, Ng S, Hyun Nam K, Ke A (2017): "How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration." Nature, 550, 137-141. doi: 10.1038/nature24020.

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes(1-5). Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer(6-9). Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical(7-9). The conserved Cas1 and Cas2 proteins form an integrase complex consisting two distal Cas1 dimers bridged by a Cas2 dimer in the middle(6,10). The prespacer is bound by Cas1-Cas2 as a dual forked DNA, and the terminal 3'-OH of each 3'-overhang serves as an attacking nucleophile during integration(11-14). Importantly, the prespacer is preferentially integrated into the leader-proximal region of the CRISPR array(1,7,10,15), guided by the leader sequence and a pair of inverted repeats (IRs) inside the CRISPR repeat(7,15-20). Spacer integration in the most well-studied Escherichia coli Type I-E CRISPR system further relies on the bacterial Integration Host Factor (IHF)(21,22). In Type II-A CRISPR, however, Cas1-Cas2 alone integrates spacer efficiently in vitro(18); other Cas proteins (Cas9 and Csn2) play accessory roles in prespacer biogenesis(17,23). Focusing on the Enterococcus faecalis Type II-A system(24), here we report four structure snapshots of Cas1-Cas2 during spacer integration. EfaCas1-Cas2 selectively binds to a splayed 30-bp prespacer bearing 4-nt 3'-overhangs. Three molecular events take place upon encountering a target: Cas1-Cas2/prespacer first searches for half-sites stochastically, then preferentially interacts with the leader-side CRISPR repeat and catalyzes a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3'-overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework explaining the stepwise spacer integration process and the leader-proximal preference.