Repetitive DNA Reeling by the Cascade-Cas3 Complex in Nucleotide Unwinding Steps

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

Research output: Contribution to journalArticlepeer-review

40 Scopus citations

Abstract

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. Loeff et al. report on a single-molecule fluorescence analysis of the E. coli CRISPR-Cas3 protein. The Cas3 protein uses a spring-loaded unwinding mechanism, reeling the target DNA 3 bp at a time. Facilitated by slipping, Cas3 repeatedly presents its intrinsically inefficient nuclease domain with DNA substrate, which may contribute to promoting a robust immune response.

Original languageEnglish
Pages (from-to)385-394.e3
JournalMolecular Cell
Volume70
Issue number3
DOIs
StatePublished - 3 May 2018

Bibliographical note

Funding Information:
C.J. was funded by the Open Program of the Division for Earth and Life Sciences (822.02.008) and Vidi (864.14.002) of the Netherlands Organization for Scientific research. S.J.J.B. was funded by an LS6 ERC starting grant (639707) and a NWO VIDI grant (864.11.005). We would like to thank S. Bailey (Johns Hopkins University) for providing the Cas3 overexpression construct. We thank T. Blosser, T. Künne, M. Klein, and R. McKenzie for critically reading this manuscript, and we thank A.C. Haagsma, M. Klein, M. Depken, M. Szczepaniak, and J. Kerssemakers for technical support.

Funding Information:
C.J. was funded by the Open Program of the Division for Earth and Life Sciences (822.02.008) and Vidi (864.14.002) of the Netherlands Organization for Scientific research. S.J.J.B. was funded by an LS6 ERC starting grant (639707) and a NWO VIDI grant (864.11.005). We would like to thank S. Bailey (Johns Hopkins University) for providing the Cas3 overexpression construct. We thank T. Blosser, T. K?nne, M. Klein, and R. McKenzie for critically reading this manuscript, and we thank A.C. Haagsma, M. Klein, M. Depken, M. Szczepaniak, and J. Kerssemakers for technical support.

Funding Information:
C.J. was funded by the Open Program of the Division for Earth and Life Sciences ( 822.02.008 ) and Vidi ( 864.14.002 ) of the Netherlands Organization for Scientific research . S.J.J.B. was funded by an LS6 ERC starting grant ( 639707 ) and a NWO VIDI grant ( 864.11.005 ). We would like to thank S. Bailey (Johns Hopkins University) for providing the Cas3 overexpression construct. We thank T. Blosser, T. Künne, M. Klein, and R. McKenzie for critically reading this manuscript, and we thank A.C. Haagsma, M. Klein, M. Depken, M. Szczepaniak, and J. Kerssemakers for technical support.

Publisher Copyright:
© 2018 Elsevier Inc.

Keywords

  • CRISPR
  • Cas3
  • FRET
  • adaptive
  • cascade
  • helicase
  • immunity
  • interference
  • single-molecule

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