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 Ϸ10-fold at 0.7 pN. At higher forces (Ϸ20 -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.DNA looping ͉ protein-DNA interactions ͉ single-molecule manipulation R estriction endonucleases (REases) are prokaryotic enzymes that act to ''restrict'' invasion of foreign DNA by cleaving phosphodiester bonds (1). These enzymes also serve as indispensable tools in molecular biology research and are used in procedures such as DNA cloning, fingerprinting, mapping, and sequencing (2). From the perspective of molecular biophysics, these enzymes are excellent model systems for studying basic principles of protein-DNA interactions (3, 4).The most commonly studied REases are of the type II variety, which cleave within or near specific recognition sites, usually require Mg 2ϩ ions as a cofactor, and do not hydrolyze ATP. More than 3,500 different type II REases having Ͼ200 different binding specificities have been identified (5). Of particular interest in our present study are the many unorthodox type II REases that do not cleave DNA efficiently if the template contains only one recognition site (6, 7). Efficient cleavage is only observed with templates containing two or more sites, suggesting that the active complex binds at two sites and the intervening DNA is looped out (7). This phenomenon of DNA looping is of broad importance in molecular biology and plays a role in many key processes including DNA transcription, replication, recombination, and repair (8-13). Looping of DNA by the Lac and Gal repressor proteins in Escherichia coli, for example, is well demonstrated and h...

Type III restriction enzymes communicate in 1D without looping between their target sites

“…3C and S3b). In contrast, for Type II REs which use 3D diffusive looping, strongly force-dependent kinetics is observed (27). We also show that DNA ends upstream of the sites can influence the efficiency of communication.…”

Cleavage mechanism of human Mus81–Eme1 acting on Holliday-junction structures

“…Another mechanism used by endonucleases to ensure cleavage of duplex DNA is cooperativity. In the case of type IIS restriction endonucleases, cooperativity and biological function are driven by induced dimerization of the enzyme subunits (35,36). The highly cooperative kinetic mechanism proposed for Mus81-Eme1 avoids first-strand cleavage unless second-strand cleavage is ensured.…”

“…The presence of two active sites in a single functional complex is thought to be essential for coordinated bilateral cleavage of duplex DNA in the case of restriction endonucleases (36) and Holliday junctions in the case of resolving enzymes (37)(38)(39). Several previously characterized resolvases have been shown to exist as highly stable dimeric complexes, whereas others undergo rapid subunit exchange in solution (27,28).…”

Light forces the pace: optical manipulation for biophotonics

“…One such protein is the ubiquitous restriction endonuclease, some varieties of which require two binding sites for cleavage to occur. 68 Some single molecule experiments use optical tweezers, not as a force measuring device, but rather as a tool to micromanipulate single molecules into a different reagent stream such that a chemical reaction may be fluorescently tracked. Bianco et al 69 did just such an experiment in their investigation of single-RECbcd enzymes.…”

“…Tug-of-war anchor-------protein-------nucleic acid-------microsphere RNA polymerase 49,57,61 ͑e.g., Fig. 4͒, helicase, 58 portal motor 48,59 ͑e.g., Popping anchor-------͑enzyme bound dsDNA͒-------microsphere Nucleosome 66,67 DNA restriction enzymes 68 Fluorescent tracking…”