title: Recycling of protein subunits during DNA translocation and cleavage by Type I restriction-modification enzymes.
abstract: The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5'-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can 'turnover' in vitro, i.e. whether they can catalyse translocation and cleavage events on one DNA molecule, dissociate and then re-bind a second DNA molecule. Translocation termination by both EcoKI and EcoR124I leads to HsdR dissociation from linear DNA but not from circular DNA. Following DNA cleavage, the HsdR subunits appear unable to dissociate even though the DNA is linear, suggesting a tight interaction with the cleaved product. The MTases of EcoKI and EcoAI can dissociate from DNA following either translocation or cleavage and can initiate reactions on new DNA molecules as long as free HsdR molecules are available. In contrast, the MTase of EcoR124I does not turnover and additional cleavage of circular DNA is not observed by inclusion of RecBCD, a helicase-nuclease that degrades the linear DNA product resulting from Type I cleavage. Roles for Type I restriction endonuclease subunit dynamics in restriction alleviation in the cell are discussed.
authors.affiliation: School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK.
authors.forename: Michelle
authors.initials: M
authors.lastname: Simons
authors.forename: Mark D
authors.initials: MD
authors.lastname: Szczelkun
mesh_terms: D004247:DNA / Q000378:metabolism*; D053837:DNA Cleavage; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D015253:Deoxyribonucleases, Type I Site-Specific / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism
chemical_list: D004270:DNA, Circular; D021122:Protein Subunits; D004247:DNA; C093244:DNA modification methylase EcoKI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D043211:Exodeoxyribonuclease V; D004262:DNA Restriction Enzymes; C089629:endodeoxyribonuclease EcoKI; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific