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

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

Author information

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

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.

PMID:
21699496
DOI:
10.1042/BJ20110901
[Indexed for MEDLINE]

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