Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

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

View ORCID ProfileLuuk Loeff, View ORCID ProfileStan J.J. Brouns, View ORCID ProfileChirlmin Joo
doi: https://doi.org/10.1101/207886
Luuk Loeff
1Kavli Institute of Nanoscience and Department of Bionanoscience, Delft University of Technology, Delft, The Netherlands
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Luuk Loeff
Stan J.J. Brouns
1Kavli Institute of Nanoscience and Department of Bionanoscience, Delft University of Technology, Delft, The Netherlands
2Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen, The Netherlands
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Stan J.J. Brouns
  • For correspondence: stanbrouns@gmail.com C.Joo@tudelft.nl
Chirlmin Joo
1Kavli Institute of Nanoscience and Department of Bionanoscience, Delft University of Technology, Delft, The Netherlands
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Chirlmin Joo
  • For correspondence: stanbrouns@gmail.com C.Joo@tudelft.nl
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

Abstract

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.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Back to top
PreviousNext
Posted October 24, 2017.
Download PDF

Supplementary Material

Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps
Luuk Loeff, Stan J.J. Brouns, Chirlmin Joo
bioRxiv 207886; doi: https://doi.org/10.1101/207886
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools
Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps
Luuk Loeff, Stan J.J. Brouns, Chirlmin Joo
bioRxiv 207886; doi: https://doi.org/10.1101/207886

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Biophysics
Subject Areas
All Articles
  • Animal Behavior and Cognition (4237)
  • Biochemistry (9151)
  • Bioengineering (6788)
  • Bioinformatics (24034)
  • Biophysics (12142)
  • Cancer Biology (9550)
  • Cell Biology (13798)
  • Clinical Trials (138)
  • Developmental Biology (7643)
  • Ecology (11719)
  • Epidemiology (2066)
  • Evolutionary Biology (15521)
  • Genetics (10654)
  • Genomics (14336)
  • Immunology (9495)
  • Microbiology (22870)
  • Molecular Biology (9113)
  • Neuroscience (49069)
  • Paleontology (355)
  • Pathology (1485)
  • Pharmacology and Toxicology (2572)
  • Physiology (3851)
  • Plant Biology (8340)
  • Scientific Communication and Education (1472)
  • Synthetic Biology (2299)
  • Systems Biology (6197)
  • Zoology (1302)