Keith Firman

Measuring motion on DNA by the type I restriction endonuclease EcoR124I using triplex displacement

The type I restriction enzyme EcoR124I cleaves DNA following extensive linear translocation dependent upon ATP hydrolysis. Using protein‐directed displacement of a DNA triplex, we have determined the kinetics of one‐dimensional motion without the necessity of measuring DNA or ATP hydrolysis. The triplex was pre‐formed specifically on linear DNA, 4370 bp from an EcoR124I site, and then incubated with endonuclease. Upon ATP addition, a distinct lag phase was observed before the triplex‐forming oligonucleotide was displaced with exponential kinetics. As the distance between type I and triplex sites was shortened, the lag time decreased whilst the displacement reaction remained exponential. This is indicative of processive DNA translocation followed by collision with the triplex and oligonucleotide displacement. A linear relationship between lag duration and inter‐site distance gives a translocation velocity of 400 ± 32 bp/s at 20°C. Furthermore, the data can only be explained by bi‐directional translocation. An endonuclease with only one of the two HsdR subunits responsible for motion could still catalyse translocation. The reaction is less processive, but can ‘reset’ in either direction whenever the DNA is released.

Real-time observation of DNA translocation by the type I restriction modification enzyme EcoR124I

Type I restriction enzymes bind sequence-specifically to unmodified DNA and subsequently pull the adjacent DNA toward themselves. Cleavage then occurs remotely from the recognition site. The mechanism by which these members of the superfamily 2 (SF2) of helicases translocate DNA is largely unknown. We report the first single-molecule study of DNA translocation by the type I restriction enzyme EcoR124I. Mechanochemical parameters such as the translocation rate and processivity, and their dependence on force and ATP concentration, are presented. We show that the two motor subunits of EcoR124I work independently. By using torsionally constrained DNA molecules, we found that the enzyme tracks along the helical pitch of the DNA molecule. This assay may be directly applicable to investigating the tracking of other DNA-translocating motors along their DNA templates.

Repercussions of DNA tracking by the type IC restriction endonuclease EcoR124I on linear, circular and catenated substrates

Type I restriction endonucleases such as EcoR124I cleave DNA at undefined loci, distant from their recognition sequences, by a mechanism that involves the enzyme tracking along the DNA between recognition and cleavage sites. This mechanism was examined on plasmids that carried recognition sites for EcoR124I and recombination sites for resolvase, the latter to create DNA catenanes. Supercoiled substrates with either one or two restriction sites were linearized by EcoR124I at similar rates, although the two‐site molecule underwent further cleavage more readily than the one‐site DNA. The catenane from the plasmid with one EcoR124I site, carrying the site on the smaller of the two rings, was cleaved by EcoR124I exclusively in the small ring, and this underwent multiple cleavage akin to the two‐site plasmid. Linear substrates derived from the plasmids were cleaved by EcoR124I at very slow rates. The communication between recognition and cleavage sites therefore cannot stem from random looping. Instead, it must follow the DNA contour between the sites. On a circular DNA, the translocation of non‐specific DNA past the specifically bound protein should increase negative supercoiling in one domain and decrease it in the other. The ensuing topological barrier may be the trigger for DNA cleavage.

Protein-polymer nano-machines. Towards synthetic control of biological processes

The exploitation of natures machinery at length scales below the dimensions of a cell is an exciting challenge for biologists, chemists and physicists, while advances in our understanding of these biological motifs are now providing an opportunity to develop real single molecule devices for technological applications. Single molecule studies are already well advanced and biological molecular motors are being used to guide the design of nano-scale machines. However, controlling the specific functions of these devices in biological systems under changing conditions is difficult. In this review we describe the principles underlying the development of a molecular motor with numerous potential applications in nanotechnology and the use of specific synthetic polymers as prototypic molecular switches for control of the motor function. The molecular motor is a derivative of a TypeI Restriction-Modification (R-M) enzyme and the synthetic polymer is drawn from the class of materials that exhibit a temperature-dependent phase transition.The potential exploitation of single molecules as functional devices has been heralded as the dawn of new era in biotechnology and medicine. It is not surprising, therefore, that the efforts of numerous multidisciplinary teams [1, 2]. have been focused in attempts to develop these systems. as machines capable of functioning at the low sub-micron and nanometre length-scales [3]. However, one of the obstacles for the practical application of single molecule devices is the lack of functional control methods in biological media, under changing conditions. In this review we describe the conceptual basis for a molecular motor (a derivative of a TypeI Restriction-Modification enzyme) with numerous potential applications in nanotechnology and the use of specific synthetic polymers as prototypic molecular switches for controlling the motor function [4].

Dynamics of initiation, termination and reinitiation of DNA translocation by the motor protein EcoR124I

Type I restriction enzymes use two motors to translocate DNA before carrying out DNA cleavage. The motor function is accomplished by amino‐acid motifs typical for superfamily 2 helicases, although DNA unwinding is not observed. Using a combination of extensive single‐molecule magnetic tweezers and stopped‐flow bulk measurements, we fully characterized the (re)initiation of DNA translocation by EcoR124I. We found that the methyltransferase core unit of the enzyme loads the motor subunits onto adjacent DNA by allowing them to bind and initiate translocation. Termination of translocation occurs owing to dissociation of the motors from the core unit. Reinitiation of translocation requires binding of new motors from solution. The identification and quantification of further initiation steps—ATP binding and extrusion of an initial DNA loop—allowed us to deduce a complete kinetic reinitiation scheme. The dissociation/reassociation of motors during translocation allows dynamic control of the restriction process by the availability of motors. Direct evidence that this control mechanism is relevant in vivo is provided.

High-level expression of the cloned genes encoding the subunits of and intact DNA methyltransferase, M·EcoR124

We have cloned the genes coding for the two subunits (HsdM and HsdS) of the type-I DNA methyltransferase (MTase), M.EcoR124, into the specially constructed expression vector, pJ119. These subunits have been synthesized together as an intact MTase. We have also cloned the individual subunit-encoding genes under the control of the T7 gene 10 promoter or the lacUV5 promoter. High levels of expression have been obtained in all cases. While HsdM was found to be soluble, HsdS was insoluble. However, in the presence of the co-produced HsdM subunit, HsdS was found in the soluble fraction as part of an active MTase. We have partially purified the cloned multi-subunit enzyme and shown that it is capable of DNA methylation both in vivo and in vitro.

AFM protein–protein interactions within the EcoR124I molecular motor

Dynamic Force Spectroscopy (DFS), an Atomic Force Microscopy (AFM) technique, has been used to investigate the interaction between the HsdR subunit and the core methylase (MTase) of the Type I Restriction-Modification (R-M) enzyme EcoR124I. Such systems are of interest in bionanotechnology owing to their ability to translocate DNA, thus acting as molecular motors. Forces between a glutathione S-transferase (GST)-HsdR(PrrI) motor subunit attached to an AFM tip using a polyethylene gycol linker and the core MTase on poly-L-lysine pre-treated mica were measured at different loading rates. In the absence of an applied force, the position of energy barrier xdiss, bond dissociation rate kdiss(0) and lifetime of the bond τ(0) were calculated to be 1.35 ± 0.17 nm, 0.16 s−1 and 6.3 s, respectively. The kdiss(0) value was a little lower than that obtained from magnetic tweezers (0.4 s−1), suggesting that the thermodynamic equilibrium may be affected by the presence of DNA. This work demonstrates that kinetic data concerning protein–protein interactions between subunits within Type I R-M enzymes are accessible via AFM. Such information is important for structure elucidation and the development of nanodevices.

Mutation in the specificity polypeptide of the type I restriction endonuclease R · EcoK that affects subunit assembly

We describe the isolation and characterization of a temperature-sensitive mutation within the hsdS gene of the type I restriction and modification system EcoK. This mutation appears to affect the ability of the HsdR subunit to interact with the HsdS subunit when forming an active endonuclease. We discuss the possibility that this mutant, together with another mutation described previously, may define a discontinuous domain, involved in protein-protein interactions, within the HsdS polypeptide.

EcoR124I: from Plasmid-Encoded Restriction-Modification System to Nanodevice

SUMMARY Plasmid R124 was first described in 1972 as being a new member of incompatibility group IncFIV, yet early physical investigations of plasmid DNA showed that this type of classification was more complex than first imagined. Throughout the history of the study of this plasmid, there have been many unexpected observations. Therefore, in this review, we describe the history of our understanding of this plasmid and the type I restriction-modification (R-M) system that it encodes, which will allow an opportunity to correct errors, or misunderstandings, that have arisen in the literature. We also describe the characterization of the R-M enzyme EcoR124I and describe the unusual properties of both type I R-M enzymes and EcoR124I in particular. As we approached the 21st century, we began to see the potential of the EcoR124I R-M enzyme as a useful molecular motor, and this leads to a description of recent work that has shown that the R-M enzyme can be used as a nanoactuator. Therefore, this is a history that takes us from a plasmid isolated from (presumably) an infected source to the potential use of the plasmid-encoded R-M enzyme in bionanotechnology.

A Synthetic Biology Project – Developing a single‐molecule device for screening drug–target interactions

This review describes a European‐funded project in the area of Synthetic Biology. The project seeks to demonstrate the application of engineering techniques and methodologies to the design and construction of a biosensor for detecting drug–target interactions at the single‐molecule level. Production of the proteins required for the system followed the principle of previously described “bioparts” concepts (a system where a database of biological parts – promoters, genes, terminators, linking tags and cleavage sequences – is used to construct novel gene assemblies) and cassette‐type assembly of gene expression systems (the concept of linking different “bioparts” to produce functional “cassettes”), but problems were quickly identified with these approaches. DNA substrates for the device were also constructed using a cassette‐system. Finally, micro‐engineering was used to build a magnetoresistive Magnetic Tweezer device for detection of single molecule DNA modifying enzymes (motors), while the possibility of constructing a Hall Effect version of this device was explored. The device is currently being used to study helicases from Plasmodium as potential targets for anti‐malarial drugs, but we also suggest other potential uses for the device.