title: Functional coupling of duplex translocation to DNA cleavage in a type I restriction enzyme.
abstract: Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.
authors.affiliation: Center for Nanobiology and Structural Biology, Institute of Microbiology and Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Csefalvay
authors.affiliation: Center for Nanobiology and Structural Biology, Institute of Microbiology and Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic.
authors.forename: Mikalai
authors.initials: M
authors.lastname: Lapkouski
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4, Czech Republic.
authors.forename: Alena
authors.initials: A
authors.lastname: Guzanova
authors.affiliation: Center for Nanobiology and Structural Biology, Institute of Microbiology and Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic.
authors.forename: Ladislav
authors.initials: L
authors.lastname: Csefalvay
authors.affiliation: Center for Nanobiology and Structural Biology, Institute of Microbiology and Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic; Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic.
authors.forename: Tatsiana
authors.initials: T
authors.lastname: Baikova
authors.affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4, Czech Republic.
authors.forename: Igor
authors.initials: I
authors.lastname: Shevelev
authors.affiliation: Center for Nanobiology and Structural Biology, Institute of Microbiology and Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic; Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic.
authors.forename: Vitali
authors.initials: V
authors.lastname: Bialevich
authors.affiliation: Center for Nanobiology and Structural Biology, Institute of Microbiology and Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic; Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic.
authors.forename: Katsiaryna
authors.initials: K
authors.lastname: Shamayeva
authors.affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4, Czech Republic; Institute of Molecular Cancer Research, University of Zürich, Wintherthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Pavel
authors.initials: P
authors.lastname: Janscak
authors.affiliation: Center for Nanobiology and Structural Biology, Institute of Microbiology and Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic; Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic.
authors.forename: Ivana
authors.initials: I
authors.lastname: Kuta Smatanova
authors.affiliation: Australian Synchrotron, 800 Blackburn Road, Clayton VIC 3168, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, VIC 3800 Australia.
authors.forename: Santosh
authors.initials: S
authors.lastname: Panjikar
authors.affiliation: Center for Nanobiology and Structural Biology, Institute of Microbiology and Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic; Chemistry Department, Princeton University, Princeton, New Jersey 08544-1009, United States of America.
authors.forename: Jannette
authors.initials: J
authors.lastname: Carey
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4, Czech Republic.
authors.forename: Marie
authors.initials: M
authors.lastname: Weiserova
authors.affiliation: Center for Nanobiology and Structural Biology, Institute of Microbiology and Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic; Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Zamek 136, CZ-373 33 Nove Hrady, Czech Republic.
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Ettrich
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D053837:DNA Cleavage; D004269:DNA, Bacterial; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D015253:Deoxyribonucleases, Type I Site-Specific; C109830:HsdR protein, E coli