title: Structural and functional analysis of the symmetrical Type I restriction endonuclease R.EcoR124INT
abstract: Type I restriction-modification (RM) systems are comprised of two multi-subunit enzymes, the methyltransferase (∼160 kDa), responsible for methylation of DNA, and the restriction endonuclease (∼400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits. An engineered RM system, EcoR124INT, based on the N-terminal domain of the specificity subunit of EcoR124I was constructed that recognises the symmetrical sequence GAAN7TTC and is active as a methyltransferase. Here, we investigate the restriction endonuclease activity of R.EcoR124INTin vitro and the subunit assembly of the multi-subunit enzyme. Finally, using small-angle neutron scattering and selective deuteration, we present a low-resolution structural model of the endonuclease and locate the motor subunits within the multi-subunit enzyme. We show that the covalent linkage between the two target recognition domains of the specificity subunit is not required for subunit assembly or enzyme activity, and discuss the implications for the evolution of Type I enzymes.
authors.affiliation: Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, United Kingdom.
authors.forename: James E
authors.initials: JE
authors.lastname: Taylor
authors.affiliation: Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, United Kingdom.
authors.forename: Anna
authors.initials: A
authors.lastname: Swiderska
authors.affiliation: Partnership for Structural Biology, Institut Laue-Langevin, Grenoble, France.|Macromolecular Structure Research Group, Keele University, Keele, Staffordshire, United Kingdom.
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Artero
authors.affiliation: Partnership for Structural Biology, Institut Laue-Langevin, Grenoble, France.
authors.forename: Philip
authors.initials: P
authors.lastname: Callow
authors.affiliation: Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, United Kingdom.
authors.forename: Geoff
authors.initials: G
authors.lastname: Kneale
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D033363:Neutron Diffraction; D011487:Protein Conformation; D015202:Protein Engineering; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D053838:Scattering, Small Angle
chemical_list: D021122:Protein Subunits; D004247:DNA; D008780:Methyltransferases; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific