DNA mimicry by proteins.

It has been discovered recently, via structural and biophysical analyses, that proteins can mimic DNA structures in order to inhibit proteins that would normally bind to DNA. Mimicry of the phosphate backbone of DNA, the hydrogen-bonding properties of the nucleotide bases and the bending and twisting of the DNA double helix are all present in the mimics discovered to date. These mimics target a range of proteins and enzymes such as DNA restriction enzymes, DNA repair enzymes, DNA gyrase and nucleosomal and nucleoid-associated proteins. The unusual properties of these protein DNA mimics may provide a foundation for the design of targeted inhibitors of DNA-binding proteins.

StpA protein from Escherichia coli condenses supercoiled DNA in preference to linear DNA and protects it from digestion by DNase I and EcoKI.

The nucleoid-associated protein, StpA, of Escherichia coli binds non-specifically to double-stranded DNA (dsDNA) and apparently forms bridges between adjacent segments of the DNA. Such a coating of protein on the DNA would be expected to hinder the action of nucleases. We demonstrate that StpA binding hinders dsDNA cleavage by both the non-specific endonuclease, DNase I, and by the site-specific type I restriction endonuclease, EcoKI. It requires approximately one StpA molecule per 250-300 bp of supercoiled DNA and approximately one StpA molecule per 60-100 bp on linear DNA for strong inhibition of the nucleases. These results support the role of StpA as a nucleoid-structuring protein which binds DNA segments together. The inhibition of EcoKI, which cleaves DNA at a site remote from its initial target sequence after extensive DNA translocation driven by ATP hydrolysis, suggests that these enzymes would be unable to function on chromosomal DNA even during times of DNA damage when potentially lethal, unmodified target sites occur on the chromosome. This supports a role for nucleoid-associated proteins in restriction alleviation during times of cell stress.

Single molecule fluorescence imaging and its application to the study of DNA condensation.

Single molecule fluorescence imaging incorporated with optical tweezers and a laminar flow cell has been used to monitor the kinetic process of DNA condensation induced by spermidine. It was found that at least two steps were involved in the condensation process of the hydrodynamically-stretched linear DNA; a lag period followed by a rapid collapse of DNA. The lag time increased with the flow speed and the collapse time remained short within the range of the flow speed studied. The effect of salt concentration on the condensation process was examined, and the results suggest that the longer lag time observed in the higher salt buffer probably results from the displacement of bound cations and rearrangement of spermidine on the DNA. The flow-speed dependence of the lag time suggests that a nucleation event at the free end of the DNA, i.e. formation of a loop, may play a vital role in the kinetic process of condensation.

Unusual 2-aminopurine fluorescence from a complex of DNA and the EcoKI methyltransferase.

The methyltransferase, M.EcoKI, recognizes the DNA sequence 5'-AACNNNNNNGTGC-3' and methylates adenine at the underlined positions. DNA methylation has been shown by crystallography to occur via a base flipping mechanism and is believed to be a general mechanism for all methyltransferases. If no structure is available, the fluorescence of 2-aminopurine is often used as a signal for base flipping as it shows enhanced fluorescence when its environment is perturbed. We find that 2-aminopurine gives enhanced fluorescence emission not only when it is placed at the M.EcoKI methylation sites but also at a location adjacent to the target adenine. Thus it appears that 2-aminopurine fluorescence intensity is not a clear indicator of base flipping but is a more general measure of DNA distortion. Upon addition of the cofactor S-adenosyl-methionine to the M.EcoKI:DNA complex, the 2-aminopurine fluorescence changes to that of a new species showing excitation at 345 nm and emission at 450 nm. This change requires a fully active enzyme, the correct cofactor and the 2-aminopurine located at the methylation site. However, the new fluorescent species is not a covalently modified form of 2-aminopurine and we suggest that it represents a hitherto undetected physicochemical form of 2-aminopurine.

Interaction of the ocr gene 0.3 protein of bacteriophage T7 with EcoKI restriction/modification enzyme.

The ocr protein, the product of gene 0.3 of bacteriophage T7, is a structural mimic of the phosphate backbone of B-form DNA. In total it mimics 22 phosphate groups over approximately 24 bp of DNA. This mimicry allows it to block DNA binding by type I DNA restriction enzymes and to inhibit these enzymes. We have determined that multiple ocr dimers can bind stoichiometrically to the archetypal type I enzyme, EcoKI. One dimer binds to the core methyltransferase and two to the complete bifunctional restriction and modification enzyme. Ocr can also bind to the component subunits of EcoKI. Binding affinity to the methyltransferase core is extremely strong with a large favourable enthalpy change and an unfavourable entropy change. This strong interaction prevents the dissociation of the methyltransferase which occurs upon dilution of the enzyme. This stabilisation arises because the interaction appears to involve virtually the entire surface area of ocr and leads to the enzyme completely wrapping around ocr.

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.

Crystallization and preliminary X-ray analysis of ocr, the product of gene 0.3 of bacteriophage T7.

Ocr, the product of gene 0.3 of bacteriophage T7, prevents the action of restriction endonucleases of the host bacteria. The amino-acid sequence of ocr has less than 20% similarity to any protein of known three-dimensional structure. Ocr has been crystallized in a number of different crystal forms and X-ray data for the seleno-L-methionine-substituted form has been collected to a resolution of 1.8 A. The presence of caesium was found to be required for good crystal growth. Anomalous X-ray data was used to identify possible positions for Se and Cs atoms in the unit cell.

Nucleoside triphosphate-dependent restriction enzymes.

The known nucleoside triphosphate-dependent restriction enzymes are hetero-oligomeric proteins that behave as molecular machines in response to their target sequences. They translocate DNA in a process dependent on the hydrolysis of a nucleoside triphosphate. For the ATP-dependent type I and type III restriction and modification systems, the collision of translocating complexes triggers hydrolysis of phosphodiester bonds in unmodified DNA to generate double-strand breaks. Type I endonucleases break the DNA at unspecified sequences remote from the target sequence, type III endonucleases at a fixed position close to the target sequence. Type I and type III restriction and modification (R-M) systems are notable for effective post-translational control of their endonuclease activity. For some type I enzymes, this control is mediated by proteolytic degradation of that subunit of the complex which is essential for DNA translocation and breakage. This control, lacking in the well-studied type II R-M systems, provides extraordinarily effective protection of resident DNA should it acquire unmodified target sequences. The only well-documented GTP-dependent restriction enzyme, McrBC, requires methylated target sequences for the initiation of phosphodiester bond cleavage.

Shape of Ocr, the gene 0.3 protein of bacteriophage T7: modeling based on light scattering experiments.

Ocr, the first protein expressed by bacteriophage T7, inhibits type Iota DNA restriction enzymes by preventing them from binding to DNA. This inhibition allows the phage to successfully infect the host. The shape of ocr is modeled on the basis of static and dynamic light scattering measurements. The static light scattering data confirm previous observations that ocr exists in solution as a dimer. The diffusion constant determined by dynamic light scattering indicates a nonspherical shape of the ocr dimer. Hydrodynamic models of ellipsoids are presented, and it is argued that ocr is best described by a prolate ellipsoid with dimensions of 10.4 nm by 2.6 nm. The size and shape predicted by this model are consistent with ocr acting as a mimic of the DNA structure bound by type Iota restriction enzymes.

Characterisation of the structure of ocr, the gene 0.3 protein of bacteriophage T7.

The product of gene 0.3 of bacteriophage T7, ocr, is a potent inhibitor of type I DNA restriction and modification enzymes. We have used biophysical methods to examine the mass, stability, shape and surface charge distribution of ocr. Ocr is a dimeric protein with hydrodynamic behaviour equivalent to a prolate ellipsoid of axial ratio 4.3 +/- 0.7:1 and mass of 27 kDa. The protein is resistant to denaturation but removal of the C-terminal region reduces stability substantially. Six amino acids, N4, D25, N43, D62, S68 and W94, are all located on the surface of the protein and N4 and S68 are also located at the interface between the two 116 amino acid monomers. Negatively charged amino acid side chains surround W94 but these side chains are not part of the highly acidic C-terminus after W94. Ocr is able to displace a short DNA duplex from the binding site of a type I enzyme with a dissociation constant of the order of 100 pM or better. These results suggest that ocr is of a suitable size and shape to effectively block the DNA binding site of a type I enzyme and has a large negatively charged patch on its surface. This charge distribution may be complementary to the charge distribution within the DNA binding site of type I DNA restriction and modification enzymes.

DNA recognition by the methyl-CpG binding domain of MeCP2.

The methyl-CpG binding domain (MBD) of the transcriptional repressor MeCP2 has been proposed to recognize a single symmetrically methylated CpG base pair via hydrophobic patches on an otherwise positively charged DNA binding surface. We have tested this binding model by analysis of mutant derivatives of the MeCP2 MBD in electrophoretic mobility shift assays complemented by NMR structural analysis. Exposed arginine side chains on the binding face, in particular Arg-111, were found to be critical for binding. Arg-111 was found to interact with the conserved aspartate side chain Asp-121, which is proposed to orientate the arginine side chain to allow specific contacts with the DNA. The conformational flexibility of the disordered B-C loop region, which forms part of the binding face, was also shown to be important. In contrast, mutation of the exposed hydrophobic side chains had a less severe effect on DNA binding. This suggests that the Arg-111 side chain may contribute to sequence-specific recognition of the CpG site rather than simply making nonspecific contacts with the phosphate backbone. The majority of missense mutations within the MBD found in the human genetic disorder Rett syndrome were shown or predicted to affect folding of the domain rather than the DNA recognition event directly.

Translocation-independent dimerization of the EcoKI endonuclease visualized by atomic force microscopy.

Bacterial type I restriction/modification systems are capable of performing multiple actions in response to the methylation pattern on their DNA recognition sequences. The enzymes making up these systems serve to protect the bacterial cells against viral infection by binding to their recognition sequences on the invading DNA and degrading it after extensive ATP-driven translocation. DNA cleavage has been thought to occur as the result of a collision between two translocating enzyme complexes. Using atomic force microscopy (AFM), we show here that EcoKI dimerizes rapidly when bound to a plasmid containing two recognition sites for the enzyme. Dimerization proceeds in the absence of ATP and is also seen with an EcoKI mutant (K477R) that is unable to translocate DNA. Only monomers are seen when the enzyme complex binds to a plasmid containing a single recognition site. Based on our results, we propose that the binding of EcoKI to specific DNA target sequences is accompanied by a conformational change that leads rapidly to dimerization. This event is followed by ATP-dependent translocation and cleavage of the DNA.

Central modules of the vaccinia virus complement control protein are not in extensive contact.

The 28.6 kDa vaccinia virus complement control protein (VCP) is an inhibitor of the complement system and has therapeutic potential. It is composed of four domains or modules and is a homologue of complement receptor 1 (CR1) and other mammalian regulators of complement activation. A key aspect to structure-function relationships in these proteins is the extent of intramolecular module-module interactions, since these dictate the overall shape and flexibility of the molecules. A protein fragment (VCP approximately 2,3) encompassing modules 2 and 3 of VCP was over-expressed in Pichia pastoris. Ultracentrifugation showed that VCP approximately 2,3 is highly asymmetric with an axial ratio of 5.3:1, which is consistent with an end-to-end arrangement of the two modules. NMR spectroscopy, differential scanning calorimetry, CD and intrinsic tryptophan fluorescence were used to monitor unfolding of VCP approximately 2,3. Experiments performed over a range of temperatures and concentrations of guanidinium chloride revealed that module 2 unfolds under milder conditions than, and independently of, module 3. Unfolding of module 2 is not associated with extensive changes in amide (15)N and (1)H chemical shifts of module 3, implying that the modules do not form an extensive intermodular interface. Results obtained in this work for VCP approximately 2,3 are compared with those obtained in a study of CR1 modules 15-17 [Kirkitadze, Krych, Uhrin, Dryden, Smith, Cooper, Wang, Hauhart, Atkinson and Barlow (1999) Biochemistry 38, 7019-7031].

Co-operativity between modules within a C3b-binding site of complement receptor type 1.

Complement receptor type 1 (CR1) has 30 modules in its extracellular portion. An understanding of structure-function relationships within CR1 is being assembled gradually from studies of overlapping protein fragments. A CR1 fragment corresponding to modules 16 and 17 was expressed recombinantly as a non-glycosylated protein and its stability and unfolding characteristics studied using biophysical techniques. The results were compared with data collected previously on a CR1 fragment encompassing modules 15, 16 and 17 which together constitute a C3b-binding site (Kirkitadze, M.D., Krych, M., Uhrin, D. , Dryden, D.T.F., Smith, B.O., Wang, X., Hauhart, R., Atkinson, J.P. and Barlow, P.N. (1999) Biochemistry 38, 7019-7031). Modules within CR1 were found to co-operate during unfolding. The folding, stability and flexibility of this protein is therefore likely to be a complex function, and not just the sum, of contributions from individual modules.

On the structure and operation of type I DNA restriction enzymes.

Type I DNA restriction enzymes are large, molecular machines possessing DNA methyltransferase, ATPase, DNA translocase and endonuclease activities. The ATPase, DNA translocase and endonuclease activities are specified by the restriction (R) subunit of the enzyme. We demonstrate that the R subunit of the Eco KI type I restriction enzyme comprises several different functional domains. An N-terminal domain contains an amino acid motif identical with that forming the catalytic site in simple restriction endonucleases, and changes within this motif lead to a loss of nuclease activity and abolish the restriction reaction. The central part of the R subunit contains amino acid sequences characteristic of DNA helicases. We demonstrate, using limited proteolysis of this subunit, that the helicase motifs are contained in two domains. Secondary structure prediction of these domains suggests a structure that is the same as the catalytic domains of DNA helicases of known structure. The C-terminal region of the R subunit can be removed by elastase treatment leaving a large fragment, stable in the presence of ATP, which can no longer bind to the other subunits of Eco KI suggesting that this domain is required for protein assembly. Considering these results and previous models of the methyltransferase part of these enzymes, a structural and operational model of a type I DNA restriction enzyme is presented.

Independently melting modules and highly structured intermodular junctions within complement receptor type 1.

A segment of complement receptor type 1 (CR1) corresponding to modules 15-17 was overexpressed as a functionally active recombinant protein with N-glycosylation sites ablated by mutagenesis (referred to as CR1 approximately 15-17(-)). A protein consisting of modules 15 and 16 and another corresponding to module 16 were also overexpressed. Comparison of heteronuclear nuclear magnetic resonance (NMR) spectra for the single, double, and triple module fragments indicated that module 16 makes more extensive contacts with module 15 than with module 17. A combination of NMR, differential scanning calorimetry, circular dichroism, and tryptophan-derived fluorescence indicated a complex unfolding pathway for CR1 approximately 15-17(-). As temperature or denaturant concentration was increased, the 16-17 junction appeared to melt first, followed by the 15-16 junction, and module 17 itself; finally, modules 15 and 16 became denatured. Modules 15 and 16 adopted an intermediate state prior to total denaturation. These results are compared with a previously published study [Clark, N. S., Dodd, I, Mossakowska, D. E., Smith, R. A. G., and Gore, M. G. (1996) Protein Eng. 9, 877-884] on a fragment consisting of the N-terminal three CR1 modules which appeared to melt as a single unit.

Localization of a protein-DNA interface by random mutagenesis.

The type I restriction and modification enzymes do not possess obvious DNA-binding motifs within their target recognition domains (TRDs) of 150-180 amino acids. To identify residues involved in DNA recognition, changes were made in the amino-TRD of EcoKI by random mutagenesis. Most of the 101 substitutions affecting 79 residues had no effect on the phenotype. Changes at only seven positions caused the loss of restriction and modification activities. The seven residues identified by mutation are not randomly distributed throughout the primary sequence of the TRD: five are within the interval between residues 80 and 110. Sequence analyses have led to the suggestion that the TRDs of type I restriction enzymes include a tertiary structure similar to the TRD of the HhaI methyltransferase, and to a model for a DNA-protein interface in EcoKI. In this model, the residues within the interval identified by the five mutations are close to the protein-DNA interface. Three additional residues close to the DNA in the model were changed; each substitution impaired both activities. Proteins from twelve mutants were purified: six from mutants with partial or wild-type activity and six from mutants lacking activity. There is a strong correlation between phenotype and DNA-binding affinity, as determined by fluorescence anisotropy.

The DNA binding characteristics of the trimeric EcoKI methyltransferase and its partially assembled dimeric form determined by fluorescence polarisation and DNA footprinting.

The type I DNA restriction and modification systems of enteric bacteria display several enzymatic activities due to their oligomeric structure. Partially assembled forms of the EcoKI enzyme from E. coli K12 can display specific DNA binding properties and modification methyltransferase activity. The heterodimer of one specificity (S) subunit and one modification (M) subunit can only bind DNA whereas the addition of a second modification subunit to form M2S1 also confers methyltransferase activity. We have examined the DNA binding specificity of M1S1 and M2S1 using the change in fluorescence anisotropy which occurs on binding of a DNA probe labelled with a hexachlorofluorescein fluorophore. The dimer has much weaker affinity for the EcoKI target sequence than the trimer and slightly less ability to discriminate against other DNA sequences. Binding of both proteins is strongly dependent on salt concentration. The fluorescence results compare favourably with those obtained with the gel retardation method. DNA footprinting using exonucleaseIII and DNaseI, and methylation interference show no asymmetry, with both DNA strands being protected by the dimer and the trimer. This indicates that the dimer is a mixture of the two possible forms, M1S1 and S1M1. The dimer has a footprint on the DNA substrate of the same length as the trimer implying that the modification subunits are located on either side of the DNA helical axis rather than lying along the helical axis.