title: High pressure activation of the Mrr restriction endonuclease in Escherichia coli involves tetramer dissociation.
abstract: A sub-lethal hydrostatic pressure (HP) shock of ∼100 MPa elicits a RecA-dependent DNA damage (SOS) response in Escherichia coli K-12, despite the fact that pressure cannot compromise the covalent integrity of DNA. Prior screens for HP resistance identified Mrr (Methylated adenine Recognition and Restriction), a Type IV restriction endonuclease (REase), as instigator for this enigmatic HP-induced SOS response. Type IV REases tend to target modified DNA sites, and E. coli Mrr activity was previously shown to be elicited by expression of the foreign M.HhaII Type II methytransferase (MTase), as well. Here we measured the concentration and stoichiometry of a functional GFP-Mrr fusion protein using in vivo fluorescence fluctuation microscopy. Our results demonstrate that Mrr is a tetramer in unstressed cells, but shifts to a dimer after HP shock or co-expression with M.HhaII. Based on the differences in reversibility of tetramer dissociation observed for wild-type GFP-Mrr and a catalytic mutant upon HP shock compared to M.HhaII expression, we propose a model by which (i) HP triggers Mrr activity by directly pushing inactive Mrr tetramers to dissociate into active Mrr dimers, while (ii) M.HhaII triggers Mrr activity by creating high affinity target sites on the chromosome, which pull the equilibrium from inactive tetrameric Mrr toward active dimer.
authors.affiliation: Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.|Centre de Biochimie Structurale, CNRS UMR5048, INSERM U1054, Université Montpellier, 34000 Montpellier, France.
authors.forename: Anaïs C
authors.initials: AC
authors.lastname: Bourges
authors.affiliation: Department of Microbial and Molecular Systems, Laboratory of Food Microbiology, KU Leuven, B-3001 Leuven, Belgium.
authors.forename: Oscar E
authors.initials: OE
authors.lastname: Torres Montaguth
authors.affiliation: Department of Microbial and Molecular Systems, Laboratory of Food Microbiology, KU Leuven, B-3001 Leuven, Belgium.
authors.forename: Anirban
authors.initials: A
authors.lastname: Ghosh
authors.affiliation: Department of Microbial and Molecular Systems, Laboratory of Food Microbiology, KU Leuven, B-3001 Leuven, Belgium.
authors.forename: Wubishet M
authors.initials: WM
authors.lastname: Tadesse
authors.affiliation: Centre de Biochimie Structurale, CNRS UMR5048, INSERM U1054, Université Montpellier, 34000 Montpellier, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Declerck
authors.affiliation: Department of Microbial and Molecular Systems, Laboratory of Food Microbiology, KU Leuven, B-3001 Leuven, Belgium.
authors.forename: Abram
authors.initials: A
authors.lastname: Aertsen
authors.affiliation: Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Royer
mesh_terms: D055162:Biocatalysis; D002850:Chromatography, Gel; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004789:Enzyme Activation; D048168:Escherichia coli K12 / Q000378:metabolism*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011312:Pressure; D055503:Protein Multimerization; D013312:Stress, Physiological
chemical_list: D050505:Mutant Proteins; D049452:Green Fluorescent Proteins; D004262:DNA Restriction Enzymes