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

Jamieson Leyland Howard , Stephane Delmas , Ivana Ivancic-Bace , Edward L Bolt (1)

1 Biomedical Sciences

CRISPR/Cas is a nucleic acid processing system in bacteria and archaea that interacts with mobile genetic elements. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) DNA and RNA sequences are processed by Cas (CRISPR-associated) 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. 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 abolished Cas3 R-loop formation and instead powered Cas3 helicase unwinding of the invading RNA strand of a model R-loop substrate. R-loop formation by Cas3 required magnesium as a co-factor and was inactivated by mutagenesis of a conserved amino acid motif. Cells expressing mutant Cas3 protein were more sensitive to plaque formation by phage lambda vir. A complex of CasABCDE ("Cascade") also promoted R-loop formation and we discuss possible overlapping roles of Cas3 and Cascade in E. coli, and apparently antagonistic roles of Cas3 catalysing RNA-DNA annealing and ATP-dependent helicase unwinding.

Mots clés: Life Sciences

Jamieson Leyland Howard, Stephane Delmas, Ivana Ivancic-Bace, Edward L Bolt. Helicase dissociation and annealing of RNA-DNA hybrids by Escherichia coli Cas3 protein.. Biochemical Journal, 2011, 439 (1), pp.85-95. ⟨10.1042/BJ20110901⟩. ⟨hal-00628671⟩