Molecular Cell
Volume 70, Issue 3, 3 May 2018, Pages 385-394.e3
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Article
Repetitive DNA Reeling by the Cascade-Cas3 Complex in Nucleotide Unwinding Steps

https://doi.org/10.1016/j.molcel.2018.03.031Get rights and content
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Highlights

  • Cas3 reels target DNA in spring-loaded bursts 3 bp at a time

  • The 3-bp bursts consist of a kinetic unwinding step size of 1 nt

  • The nuclease domain of Cas3 is intrinsically inefficient in cleaving DNA

  • The helicase domain repeatedly presents target DNA to the inefficient nuclease domain.

Summary

CRISPR-Cas provides RNA-guided adaptive immunity against invading genetic elements. Interference in type I systems relies on the RNA-guided Cascade complex for target DNA recognition and the 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 Förster resonance energy transfer (FRET) to probe the helicase activity with high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling the DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of 3 bp, which underlie three successive 1-nt unwinding events. Reeling is highly repetitive, allowing Cas3 to repeatedly present its inefficient nuclease domain with single-strand DNA (ssDNA) substrate. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure controlled degradation of target DNA only.

Keywords

CRISPR
cascade
Cas3
single-molecule
FRET
helicase
adaptive
immunity
interference

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