Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex

Robert D. Fagerlund, Max E. Wilkinson, Oleg Klykov, Arjan Barendregt, F.G. Pearce, Sebastian N. Kieper, Howard W.R. Maxwell, Angela Capolupo, Albert J.R. Heck, Kurt L. Krause, Mihnea Bostina, Richard A. Scheltema, Raymond H.J. Staals, Peter C. Fineran*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

55 Citations (Scopus)

Abstract

CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas14-Cas2-32 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1-Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1-Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3′-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1-Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1-Cas2-3 adaptation complexes and their integration mechanism.

Original languageEnglish
Pages (from-to)E5122-E5128
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number26
DOIs
Publication statusPublished - 27 Jun 2017
Externally publishedYes

Keywords

  • Crispr-cas
  • Horizontal gene transfer
  • Mass spectrometry
  • Phage resistance
  • Spacer acquisition

Fingerprint Dive into the research topics of 'Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex'. Together they form a unique fingerprint.

Cite this