Helicase dissociation and annealing of RNA-DNA hybrids by Escherichia coli Cas3 protein

Biochem J. 2011 Oct 1;439(1):85-95. doi: 10.1042/BJ20110901.


CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) is a nucleic acid processing system in bacteria and archaea that interacts with mobile genetic elements. CRISPR DNA and RNA sequences are processed by Cas proteins: in Escherichia coli K-12, one CRISPR locus links to eight cas genes (cas1, 2, 3 and casABCDE), whose protein products promote protection against phage. In the present paper, we report that purified E. coli Cas3 catalyses ATP-independent annealing of RNA with DNA forming R-loops, hybrids of RNA base-paired into duplex DNA. ATP abolishes Cas3 R-loop formation and instead powers Cas3 helicase unwinding of the invading RNA strand of a model R-loop substrate. R-loop formation by Cas3 requires magnesium as a co-factor and is inactivated by mutagenesis of a conserved amino acid motif. Cells expressing the mutant Cas3 protein are more sensitive to plaque formation by the phage λvir. A complex of CasABCDE ('Cascade') also promotes R-loop formation and we discuss possible overlapping roles of Cas3 and Cascade in E. coli, and the apparently antagonistic roles of Cas3 catalysing RNA-DNA annealing and ATP-dependent helicase unwinding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism*
  • Escherichia coli K12 / genetics
  • Escherichia coli K12 / metabolism*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Magnesium / metabolism
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism*


  • DNA, Bacterial
  • Escherichia coli Proteins
  • RNA, Bacterial
  • DNA Helicases
  • Magnesium