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Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps

By Luuk Loeff, Stan J.J. Brouns, Chirlmin Joo

Posted 24 Oct 2017
bioRxiv DOI: 10.1101/207886 (published DOI: 10.1016/j.molcel.2018.03.031)

CRISPR-Cas loci provide an RNA-guided adaptive immune system against invading genetic elements. Interference in type I systems relies on the RNA-guided surveillance complex Cascade for target DNA recognition and the trans-acting Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here we employed single-molecule FRET to probe the helicase activity with a high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling in the target DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of three base pairs, that each underlie three successive 1-nt unwinding events. Reeling is highly repetitive, compensating for inefficient nicking activity of the nuclease domain. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure well controlled degradation of target DNA only.

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