title: Subunit assembly modulates the activities of the Type III restriction-modification enzyme PstII in vitro.
abstract: We demonstrate that, like other Type III restriction endonuclease, PstII does not turnover such that a DNA substrate is only fully cleaved at a Res2Mod2-to-site ratio of approximately 1:1. However, unlike other Type III enzymes, the cleavage rate profiles varied with protein concentration: using 5 nM DNA and 25 nM PstII, approximately half of the DNA was cut at a fast rate while the remainder was cut 24 times more slowly; in comparison, with 100 nM PstII cleavage occurs at a single fast rate. The inclusion of the methyl donor S-adenosyl methionine does not alter the rates with 100 nM PstII but with 25 nM PstII the reaction stopped after completion of the initial fast cleavage phase owing to methylation. Concentration-dependent rates were also observed in methylation assays: at 100 nM PstII, a single slow rate was measured while at lower PstII concentrations both fast and slow rates were measured. We propose a model in which the intact Res2Mod2 complex favoured at high PstII concentrations is a fast endonuclease/slow methyltransferase while the various subassemblies which coexist at lower concentrations are fast methyltransferases. A potential role for disassembly in control of restriction activity in vivo is discussed.
authors.affiliation: DNA-Protein Interactions Unit, Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Alice
authors.initials: A
authors.lastname: Sears
authors.forename: Mark D
authors.initials: MD
authors.lastname: Szczelkun
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000378:metabolism*; D015263:Deoxyribonucleases, Type III Site-Specific / Q000378:metabolism*; D007700:Kinetics; D017830:Octoxynol / Q000494:pharmacology; D021122:Protein Subunits / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D021122:Protein Subunits; D017830:Octoxynol; D004247:DNA; D015254:DNA Modification Methylases; D015263:Deoxyribonucleases, Type III Site-Specific