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.

Australian Synchrotron 800 Blackburn Road Clayton VIC 3168 Australia; Department of Biochemistry and Molecular Biology Monash University Clayton Campus Melbourne VIC 3800 Australia

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

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

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

Institute of Microbiology Academy of Sciences of the Czech Republic Vídeňská 1083 142 20 Praha 4 Czech Republic

Institute of Molecular Genetics Academy of Sciences of the Czech Republic Vídeňská 1083 142 20 Praha 4 Czech Republic

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

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Broadness and specificity: ArdB, ArdA, and Ocr against various restriction-modification systems

Crystal structure of a novel domain of the motor subunit of the Type I restriction enzyme EcoR124 involved in complex assembly and DNA binding

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