title: Tension-dependent DNA cleavage by restriction endonucleases: two-site enzymes are "switched off" at low force.
abstract: DNA looping occurs in many important protein-DNA interactions, including those regulating replication, transcription, and recombination. Recent theoretical studies predict that tension of only a few piconewtons acting on DNA would almost completely inhibit DNA looping. Here, we study restriction endonucleases that require interaction at two separated sites for efficient cleavage. Using optical tweezers we measured the dependence of cleavage activity on DNA tension with 15 known or suspected two-site enzymes (BfiI, BpmI, BsgI, BspMI, Cfr9I, Cfr10I, Eco57I, EcoRII, FokI, HpaII, MboII, NarI, SacII, Sau3AI, and SgrAI) and six one-site enzymes (BamHI, EcoRI, EcoRV, HaeIII, HindIII, and DNaseI). All of the one-site enzymes were virtually unaffected by 5 pN of tension, whereas all of the two-site enzymes were completely inhibited. These enzymes thus constitute a remarkable example of a tension sensing "molecular switch." A detailed study of one enzyme, Sau3AI, indicated that the activity decreased exponentially with tension and the decrease was approximately 10-fold at 0.7 pN. At higher forces (approximately 20-40 pN) cleavage by the one-site enzymes EcoRV and HaeIII was partly inhibited and cleavage by HindIII was enhanced, whereas BamHI, EcoRI, and DNaseI were largely unaffected. These findings correlate with structural data showing that EcoRV bends DNA sharply, whereas BamHI, EcoRI, and DNaseI do not. Thus, DNA-directed enzyme activity involving either DNA looping or bending can be modulated by tension, a mechanism that could facilitate mechanosensory transduction in vivo.
authors.affiliation: Department of Physics, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Gemmen
authors.forename: Rachel
authors.initials: R
authors.lastname: Millin
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Smith
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D008863:Microspheres; D009690:Nucleic Acid Conformation; D013379:Substrate Specificity
chemical_list: D004247:DNA; D004262:DNA Restriction Enzymes