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

Author:

Howard Jamieson A. L.1,Delmas Stephane2,Ivančić-Baće Ivana3,Bolt Edward L.1

Affiliation:

1. School of Biomedical Sciences, The University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, U.K.

2. School of Biology, The University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, U.K.

3. Department of Molecular Biology, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia

Abstract

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.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

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