Figure 1. (a) Assaying for a restriction–modification (R-M) system in a cell-free extract. Serial dilutions were prepared from an extract of Escherichia coli cells that contained the EcoRV R-M system and an aliquot from each dilution was added to phage DNA (0.5 g) in an appropriate reaction buffer. The reactions were incubated at 37°C for 30 min and the DNA was then analysed by electrophoresis through agarose. Undiluted cell extract (left-hand lane) results in cleavage of DNA at a series of discrete sites and at many additional sites, as judged by the smearing of the bands; the latter is due to nonspecific nucleases. The moderately-diluted extracts (central lanes) produce clearly defined fragments, indicating the presence of a sequence-specific endonuclease. At high dilutions (right-hand lanes), the amount of restriction enzyme present is no longer sufficient to cleave all (or any) of the DNA. (b) Kinetic analysis of DNA cleavage by a restriction enzyme. A sample of purified EcoRV endonuclease was added to the supercoiled form of a plasmid that has one EcoRV recognition site, in reaction buffer containing magnesium chloride. At various times after the addition of the enzyme, aliquots were removed from the mixture and mixed immediately with ethylenediaminetetraacetic acid (EDTA). The DNA in each aliquot was then analysed by electrophoresis through agarose (increasing reaction times, from left to right across the gel). With a supercoiled substrate, cleavage of one strand converts the DNA to its open circle form, whereas cleavage of both strands at the same site yields linear DNA. These forms have different electrophoretic mobilities, as indicated by S.C., O.C. and Linear on the left of the gel. To determine the kinetics of the reaction, radiolabelled DNA is used so that the amounts of each form of DNA can be measured at each time point sampled.

Figure 2. Spontaneous generation of new specificities in type I restriction–modification systems. Only the specificity (S) subunits are shown, represented in simplified form to highlight the interactions of the separate DNA-binding domains with the recognition half-sites (bold sequences). (a) StyLTIII and StySPI S subunits, shown in different colours, yield recombinant forms that recognize hybrid sites. (b) The switch between two copies of a repeated amino acid sequence in the S subunit of EcoR124I to three copies in EcoR124II (depicted as either two or three blocks in the region between DNA-binding domains) alters the length of the nonspecific spacer between recognition half-sites.

Figure 3. Crystal structures of type II R-M enzymes with their DNA recognition sites. The two DNA strands are depicted in grey and pink, respectively, and are shown in space-filling form (except for (b)). (a) The EcoRV endonuclease dimer bound to its recognition site. The two protein subunits are indicated by blue and green ribbons. (b) The structure of the specific DNA bound to the EcoRV endonuclease (a) is shown here in stick form, rotated 90° about the vertical axis relative to the view in (a). The protein is not shown here in order to reveal the bend in the DNA induced by the binding of EcoRV. (c) The FokI endonuclease monomer bound to its recognition site. The catalytic domain of FokI is shown in blue and its DNA recognition domain in green. The white arrowheads mark the positions where FokI cleaves each strand of the DNA. (d) The HhaI methyltransferase monomer bound to its recognition site. The target cytosine has been flipped out of the DNA helix to a position in the active site of the enzyme where it is methylated. M.HhaI is shown as a green ribbon, while the AdoMet cofactor (converted to S-adenosyl-l-homocysteine when it donated the methyl group) is in blue.

Figure 4. Model for the mechanism of DNA cleavage by type III restriction–modification systems. Type III enzymes bind DNA at their asymmetric recognition sequences (indicated by half-arrows) via the Mod subunit. The hydrolysis of adenosine trisphosphate (ATP) is then used to translocate DNA past the enzyme in one direction only. When two enzymes are bound to two sites in inverted orientation, the enzymes gradually draw towards one another. Restriction occurs when the two enzymes collide. Cleavage by the Res subunit takes place ~25 bp downstream of the recognition site (indicated by scissors).