David T. F. Dryden

Allostery without conformational change

A general model is presented whereby lignand-induced changes in protein dynamics could produce allosteric communication between distinct binding sites, even in the absence of a macromolecular conformational change. Theoretical analysis, based on the statistical thermodynamics of ligand binding, shows that cooperative interaction free energies amounting to several kJ · mol-1 may be generated by this means. The effect arises out of the possible changes in frequencies and amplitudes of macromolecular thermal fluctuations in response to ligand attachment, and can involve all forms of dynamic behaviour, ranging from highly correlated, low-frequency normal mode vibrations to random local anharmonic motions of individual atoms or groups. Dynamic allostery of this form is primarily an entropy effect, and we derive approximate expressions which might allow the magnitude of the interaction in real systems to be calculated directly from experimental observations such as changes in normal mode frequencies and mean-square atomic displacements. Long-range influence of kinetic processes at different sites might also be mediated by a similar mechanism. We suggest that proteins and other biological macromolecules may have evolved to take functional advantage not only of mean conformational states but also of the inevitable thermal fluctuations about the mean.

Structure of Ocr from Bacteriophage T7, a Protein that Mimics B-Form DNA

We have solved, by X-ray crystallography to a resolution of 1.8 A, the structure of a protein capable of mimicking approximately 20 base pairs of B-form DNA. This ocr protein, encoded by gene 0.3 of bacteriophage T7, mimics the size and shape of a bent DNA molecule and the arrangement of negative charges along the phosphate backbone of B-form DNA. We also demonstrate that ocr is an efficient inhibitor in vivo of all known families of the complex type I DNA restriction enzymes. Using atomic force microscopy, we have also observed that type I enzymes induce a bend in DNA of similar magnitude to the bend in the ocr molecule. This first structure of an antirestriction protein demonstrates the construction of structural mimetics of long segments of B-form DNA.

Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping

Many DNA-modifying enzymes act in a manner that requires communication between two noncontiguous DNA sites. These sites can be brought into contact either by a diffusion-mediated chance interaction between enzymes bound at the two sites, or by active translocation of the intervening DNA by a site-bound enzyme. EcoP15I, a type III restriction enzyme, needs to interact with two recognition sites separated by up to 3,500 bp before it can cleave DNA. Here, we have studied the behavior of EcoP15I, using a novel fast-scan atomic force microscope, which uses a miniaturized cantilever and scan stage to reduce the mechanical response time of the cantilever and to prevent the onset of resonant motion at high scan speeds. With this instrument, we were able to achieve scan rates of up to 10 frames per s under fluid. The improved time resolution allowed us to image EcoP15I in real time at scan rates of 1–3 frames per s. EcoP15I translocated DNA in an ATP-dependent manner, at a rate of 79 ± 33 bp/s. The accumulation of supercoiling, as a consequence of movement of EcoP15I along the DNA, could also be observed. EcoP15I bound to its recognition site was also seen to make nonspecific contacts with other DNA sites, thus forming DNA loops and reducing the distance between the two recognition sites. On the basis of our results, we conclude that EcoP15I uses two distinct mechanisms to communicate between two recognition sites: diffusive DNA loop formation and ATPase-driven translocation of the intervening DNA contour.

Highlights of the DNA cutters: a short history of the restriction enzymes

In the early 1950’s, ‘host-controlled variation in bacterial viruses’ was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine.

Type I restriction enzymes and their relatives

Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction–modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes.

Direct observation of DNA translocation and cleavage by the EcoKI endonuclease using atomic force microscopy

Direct observation of DNA translocation and cleavage by the Eco KI endonuclease using atomic force microscopy

A mutational analysis of the two motifs common to adenine methyltransferases.

All methyltransferases that use S‐adenosyl methionine as the methyl group donor contain a sequence similar to (D/E/S)XFXGXG which has been postulated to form part of the cofactor binding site. In N6‐adenine DNA methyltransferases there is a second motif, (D/N)PP(Y/F), which has been proposed to play a role similar to the catalytically essential PC motif conserved in all C5‐cytosine DNA methyltransferases. We have made a series of amino acid changes in these two motifs in the EcoKI N6‐adenine DNA methyltransferase. The mutant enzymes have been purified to homogeneity and characterized by physical biochemical methods. The first G is the most conserved residue in motif I. Changing this G to D completely abolished S‐adenosyl methionine binding, but left enzyme structure and DNA target recognition unaltered, thus documenting the S‐adenosyl methionine binding function of motif I in N6‐adenine methyltransferases. Substitution of the N with D, or F with either G or C, in motif II abolished enzyme activity, but left S‐adenosyl methionine and DNA binding unaltered. Changes of F to Y or W resulted in partial enzyme activity, implying that an aromatic residue is important for methylation. The substitution of W for F greatly enhanced UV‐induced cross‐linking between the enzyme and S‐adenosyl methionine, suggesting that the aromatic residue is close in space to the methyl‐group donor.

Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes

DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 Å resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (∼100 ps) decay component and the large increase in the amplitude of the long (∼10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA–enzyme complexes that cannot be discerned from the present X-ray structures.

Extensive DNA mimicry by the ArdA anti-restriction protein and its role in the spread of antibiotic resistance

The ardA gene, found in many prokaryotes including important pathogenic species, allows associated mobile genetic elements to evade the ubiquitous Type I DNA restriction systems and thereby assist the spread of resistance genes in bacterial populations. As such, ardA contributes to a major healthcare problem. We have solved the structure of the ArdA protein from the conjugative transposon Tn916 and find that it has a novel extremely elongated curved cylindrical structure with defined helical grooves. The high density of aspartate and glutamate residues on the surface follow a helical pattern and the whole protein mimics a 42-base pair stretch of B-form DNA making ArdA by far the largest DNA mimic known. Each monomer of this dimeric structure comprises three alpha–beta domains, each with a different fold. These domains have the same fold as previously determined proteins possessing entirely different functions. This DNA mimicry explains how ArdA can bind and inhibit the Type I restriction enzymes and we demonstrate that 6 different ardA from pathogenic bacteria can function in Escherichia coli hosting a range of different Type I restriction systems.

DNA looping and translocation provide an optimal cleavage mechanism for the type III restriction enzymes

EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site‐bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I–DNA pre‐cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site‐bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for restriction by type III restriction enzymes and highlight the similarities between this and other classes of restriction enzymes.