Many bacterial genomes encode multiple metal-sensing ArsR-SmtB transcriptional repressors. There is interest in understanding and predicting their metal specificities. Here we analyse two arsR-smtB genes, ydeT and yozA (now aseR and czrA) from Bacillus subtilis. Purified AseR and CzrA formed complexes in gel-retardation and fluorescence-anisotropy assays with fragments of promoters that were derepressed in DeltaaseR and DeltaczrA cells. Candidate (i) partly thiolate, alpha3-helix (for AseR) and (ii) tetrahedral, non-thiolate, alpha5-helix (for CzrA) metal binding sites were predicted then tested in vitro and/or in vivo. The precedents are for such sites to sense arsenite/antimonite (alpha3) and zinc (alpha5). This correlated with the respective metal inducers of AseR and CzrA repressed promoters in B. subtilis and matched the metals that impaired formation of protein-DNA complexes in vitro. The putative sensory sites of 1024 ArsR-SmtB homologues are reported. Although AseR did not sense zinc in vivo, it bound zinc in vitro exploiting alpha3 thiols, but AseR DNA binding was not impaired by zinc. If selectivity relies on discriminatory triggering of allostery not just selective metal binding, then tight non-effector metal complexes could theoretically inhibit metal sensing. AseR remained arsenite-sensitive in equimolar zinc, while CzrA remained zinc-sensitive in equimolar arsenite in vitro. However, cupric ions did not impair CzrA-DNA complex formation but did inhibit zinc-mediated allostery in vitro and prevent zinc binding. Access to copper must be controlled in vivo to avoid formation of cupric CzrA.

The mechanism of the Escherichia coli DNA T:G mismatch endonuclease (Vsr) has been investigated using oligodeoxynucleotides substituted, at the scissile phosphate, with isomeric phosphorothioates and a 3′-phosphorothiolate. Binding and kinetic data with the phosphorothioates/phosphorothiolate indicate that the two magnesium ions, which constitute essential co-factors, are required to stabilise the extra negative charge developed on the phosphate as the transition state is formed. Additionally one of the magnesium ions serves to activate the leaving group (the non-bridging 3′-oxygen atom of the scissile phosphate) during the hydrolysis reaction. Stereochemical analysis, using the Rp phosphorothioate isomer, indicates that Vsr carries out a hydrolytic reaction with inversion of stereochemistry at phosphorus, compatible with an in-line attack of water and a pentacovalent transition state with trigonal bipyramidal geometry. In conjunction with structures of Vsr bound to its products, these data allow the reconstruction of the enzyme–substrate complex and a comprehensive description of the hydrolysis mechanism.

Oligonucleotides have been synthesized on hydrogen-terminated Si(111) and porous silicon using surface hydrosilation of difunctional molecules (1,(omega)-dimethoxytritylundecenol) to produce a monolayer bearing suitable reactive groups to allow automated solid-phase DNA synthesis. The absence of an intervening oxide enables electrochemical characterisation of the surface-bound oligonucleotides. Complementary sequences to the DNA synthesized on Si(111) undergo hybridisation at the surface and a straightforward electrochemical quantitation of the amount of synthesized DNA and its hybridisation efficiency (47%) is possible using Ru(NH3)6(3+) as a redox label. In the case of DNA synthesized in porous silicon, electron transfer (ET) between DNA and the underlying bulk semiconductor can be studied by cyclic voltammetry, however the anisotropic diffusion inside the porous layer and the large resistance of the porous silicon results in voltammograms for which thin-layer behaviour is not observed and the peak currents increase with the square root of scan rate. We interpret these voltammograms in terms of charge transport limitations in the layer of metal centres bound to the DNA inside the pores. Further evidence for this interpretation has been obtained using scanning electrochemical microscopy (SECM) to study the charge transport between redox species in films of DNA synthesized on Si(111) surfaces that are in contact with an aqueous phase. As the bulk concentration of Ru(NH3)6(3+) is reduced below about 250 microM the SECM feedback indicates that the rate of charge transport between surface-bound Ru(NH3)6(3+) exceeds that due to diffusion in the liquid phase. Electrochemical quantitation of the DNA is not possible in this situation, however we have been able to obtain independent determinations using radioassay based on 32P or UV/VIS spectrophotometry of dimethoxytrityl cation cleaved from the porous layer. In the case of the former, use of labelled complementary sequences shows an inverse relationship between the current density used to prepare the porous silicon and the amount of hybridisation. This can be interpreted in terms of the specific surface area of the porous silicon layers since the hybridisation efficiencies (ca. 40%) obtained by comparing DMT+ cleaved from sequences synthesized on the surface and then from complementary sequences after hybridisation were relatively insensitive to the current density used to prepare the layers. Our recent work has also been concerned with individual Si nanocrystals generated by breaking up porous silicon during thermal hydrosilation reactions. FTIR spectroscopy shows these particles are also coated with an organic Si-C-bonded monolayer and they form stable, non-turbid and strongly luminescent (lambdamax = 600-650 nm) dispersions in apolar solvents (L. H. Lie, M. S. Duerdin, E. M. Tuite, A. Houlton and B. R. Horrocks, J. Electroanal. Chem., 2002, 538/539, 183). The effect of carrying out synthetic reactions on the porous silicon prior to breaking up the layer is to produce instead larger, micron-scale assemblies with a nanometre scale internal structure. Micron-sized particles of porous silicon produced by breaking up the layer can be probed by confocal Raman spectroscopy using the electric field of a focused laser to trap such particles. Although these particles are also luminescent, the use of relatively long wavelength laser excitation (lambda = 785 nm) allows acquisition of Raman spectra from individual particles in the optical trap. The bulk optical phonon mode at ca. 520 cm(-1) characteristic of crystalline silicon is red-shifted and broadened providing evidence for an internal nanometre scale substructure in these micron-sized particles and we also see evidence for this mode in the colloidal suspensions of the Si nanoparticles. We propose a model for the formation of these two types of particles and briefly discuss the prospects to extend our solid-phase synthesis on porous silicon to allow the facile synthesis of luminescent Si nanocrystals bearing DNA or other biomolecules.

Archaeal family B DNA polymerases contain a specialised pocket that binds tightly to template-strand uracil, causing the stalling of DNA replication. The mechanism of this unique “template-strand proof-reading” has been studied using equilibrium binding measurements, DNA footprinting, van't Hoff analysis and calorimetry. Binding assays have shown that the polymerase preferentially binds to uracil in single as opposed to double-stranded DNA. Tightest binding is observed using primer–templates that contain uracil four bases in front of the primer–template junction, corresponding to the observed stalling position. Ethylation interference analysis of primer–templates shows that the two phosphates, immediately flanking the uracil (NpUpN), are important for binding; contacts are also made to phosphates in the primer-strand. Microcalorimetry and van't Hoff analysis have given a fuller understanding of the thermodynamic parameters involved in uracil recognition. All the results are consistent with a “read-ahead” mechanism, in which the replicating polymerase scans the template, ahead of the replication fork, for the presence of uracil and halts polymerisation on detecting this base. Post-stalling events, serving to eliminate uracil, await full elucidation.

The ferrocenyl-nucleoside, 5-ethynylferrocenyl-2'-deoxycytidine (1) has been prepared by Pd-catalyzed cross-coupling between ethynylferrocene and 5-iodo-2'-deoxycytidine and incorporated into oligonucleotides by using automated solid-phase synthesis at both silica supports (CPG) and modified single-crystal silicon electrodes. Analysis of DNA oligonucleotides prepared and cleaved from conventional solid supports confirms that the ferrocenyl-nucleoside remains intact during synthesis and deprotection and that the resulting strands may be oxidised and reduced in a chemically reversible manner. Melting curve data show that the ferrocenyl-modified oligonucleotides form duplex structures with native complementary strands. The redox potential of fully solvated ferrocenyl 12-mers, 350 mV versus SCE, was shifted by +40 mV to a more positive potential upon treatment with the complement contrary to the anticipated negative shift based on a simple electrostatic basis. Automated solid-phase methods were also used to synthesise 12-mer ferrocenyl-containing oligonucleotides directly at chemically modified silicon <111> electrodes. Hybridisation to the surface-bound ferrocenyl-DNA caused a shift in the reduction potential of +34 mV to more positive values, indicating that, even when a ferrocenyl nucleoside is contained in a film, the increased density of anions from the phosphate backbone of the complement is still dominated by other factors, for example, the hydrophobic environment of the ferrocene moiety in the duplex or changes in the ferrocene-phosphate distances. The reduction potential is shifted >100 mV after hybridisation when the aqueous electrolyte is replaced by THF/LiClO(4), a solvent of much lower dielectric constant; this is consistent with an explanation based on conformation-induced changes in ferrocene-phosphate distances.

Random mutagenesis constitutes an important approach for identifying critical regions of proteins, studying structure-function relations and developing novel proteins with desired properties. Perhaps, the most popular method is the error-prone PCR, in which mistakes are introduced into a gene, and hence a protein, during DNA polymerase-catalysed amplification cycles. Unfortunately, the relatively high fidelities of the thermostable DNA polymerases commonly used for PCR result in too few mistakes in the amplified DNA for efficient mutagenesis. In this paper, we describe mutants of the family B DNA polymerase from Pyrococcus furiosus (Pfu-Pol), with superb performance in error-prone PCR. The key amino acid changes occur in a short loop linking two long alpha-helices that comprise the 'fingers' sub-domain of the protein. This region is responsible for binding the incoming dNTPs and ensuring that only correct bases are inserted opposite the complementary base in the template strand. Mutations in the short loop, when combined with an additional mutation that abolishes the 3'-5' proof-reading exonuclease activity, convert the extremely accurate wild-type polymerase into a variant with low fidelity. The mutant Pfu-Pols can be applied in error-prone PCR, under exactly the same conditions used for standard, high-fidelity PCR with the wild-type enzyme. Large quantities of amplified product, with a high frequency of nearly indiscriminate mutations, are produced. It is anticipated that the Pfu-Pol variants will be extremely useful for the randomization of gene, and hence protein, sequences.

The EcoRV restriction endonuclease recognises palindromic GATATC sequences and cuts between the central T and dA bases in a reaction that has an absolute requirement for a divalent metal ion, physiologically Mg2+. Use has been made of base analogues, which delete hydrogen bonds between the protein and DNA (or hydrophobic interactions in the case of the 5-CH3 group of thymine), to evaluate the roles of the outer two base-pairs (GATATC) in DNA recognition. Selectivity arises at both the binding steps leading to the formation of the enzyme–DNA–metal ion ternary complex (assayed by measuring the dissociation constant in the presence of the non-reactive metal Ca2+) and the catalytic step (evaluated using single-turnover hydrolysis in the presence of Mg2+), with each protein–DNA contact contributing to recognition. With the A:T base-pair, binding was reduced by the amount expected for the simple loss of a single contact; much more severe effects were observed with the G:C base-pair, suggesting additional conformational perturbation. Most of the modified bases lowered the rate of hydrolysis; furthermore, the presence of an analogue in one strand of the duplex diminished cutting at the second, unmodified strand, indicative of communication between DNA binding and the active site. The essential metal ion Mg2+ plays a key role in mediating interactions between the DNA binding site and active centre and in many instances rescue of hydrolysis was seen with Mn2+. It is suggested that contacts between the GATATC site are required for tight binding and for the correct assembly of metal ions and bound water at the catalytic site, functions important in providing acid/base catalysis and transition state stabilisation.

DNA and alkenyl pyrrole functionalized silicon surfaces allow for the formation of silicon/polypyrrole junctions. These junctions are spatially controlled by photolithography (see Figure) and selective photo‐assisted electro‐oxidation of pyrrole. This process shows promise for use in the development of molecular‐based electronic devices, and could enable the deposition of polypyrrole analogously to metal ions in the formation of metallic wires.

Archaeal family-B DNA polymerases possess a novel uracil-sensing mechanism. A specialized pocket scans the template, ahead of the replication fork, for the presence of uracil; on encountering this base, DNA synthesis is stalled. The structural basis for uracil recognition by polymerases is described and compared with other uracil-recognizing enzymes (uridine-triphosphate pyrophophatases and uracil-DNA glycosylases). Remarkably, protein-protein interactions between all three archaeal uracil sensors are observed; possibly the enzymes co-operate to efficiently eliminate uracil from archaeal genomes.

The various surface types involved in the synthesis of DNA upon alcohol-terminated monolayers at Si(111) surfaces were probed using scanning tunneling microscopy (STM). Images of the initial hydrogen-terminated layer showed the typical step and terrace features which are retained after alkylation with 4,4'-dimethoxytrityl-1-undecenol. The method of deprotection for oligonucleotides synthesized at these monolayer films was shown to have a dramatic effect on the surface. After treatment with methylamine, rinsing with water caused considerable etching of the surface; however, this was avoided by the use of a dry ethyl acetate and ether wash. There is, little indication of DNA for surfaces covered with single stranded material as judged by STM imaging or tunneling spectroscopy experiments. However, after hybridization with the complementary strand, DNA-Si(111) substrates gave stable images that do not typically show the step and terrace features of the underlying alkyl monolayer. Instead wormlike features are seen that are interpreted as containing aligned DNA molecules lying nearly parallel to the surface.

Complementary strands and redox intercalators can be self-assembled into nanoscale structures capable of charge transfer at the electrode surface from DNA oligomers synthesized directly at covalently modified semiconductor silicon surfaces (see schematic representation).

Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway, have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here, we describe the interaction between the C5 MTase from Haemophilus haemolyticus (M.HhaI) and an oligodeoxynucleotide duplex containing 2-H pyrimidinone, an analogue often referred to as zebularine and known to give rise to high-affinity complexes with MTases. X-ray crystallography has demonstrated the formation of a covalent bond between M.HhaI and the 2-H pyrimidinone-containing oligodeoxynucleotide. This observation enables a comparison between the mechanisms of action of 2-H pyrimidinone with other mechanism-based inhibitors such as FdC. This novel complex provides a molecular explanation for the mechanism of action of the anti-cancer drug zebularine.

Ferrocenylthymidine derivatives have been prepared by Pd-catalysed cross-coupling between ethynylferrocene or vinylferrocene and 5-iodo-2'-deoxyuridine. In the latter case a mixture of trans (2a) and gem (2b) isomers was obtained. The cis-vinylferrocenyl (2c), and ethylferrocenyl (3) derivatives were obtained by catalytic hydrogenation of ethynylferrocenyl-dT (1a), and 2c respectively. Single-crystal X-ray data for 1a, the ferrocenyl-2'furano-pyrimidone 1b, and 2a show that the nucleobase is essentially co-planar with the substituted Cp ring of the metallocene. The selective reduction of the linkage between the ferrocenyl and thymidine moieties, from -C identical to C- to -CH2CH2-, causes a shift in the reduction potential of -124 mV. DFT calculations for the one-electron oxidised species indicate that the diminished conjugation reduces the spin transfer onto the bridging C2 group, but has less effect on the extent transferred to the nucleobase from the ferrocenyl group. Compound 1a was incorporated site-specifically into DNA oligonucleotides by using automated solid-phase methods. However, some interconversion of 1a-->1b occurs, even under rapid mild conditions of deprotection.

The type IIs restriction endonucleaseMboII recognizes nonsymmetrical GAAGA sites, cutting 8 (top strand) and 7 (bottom strand) bases to the right. Gel retardation showed that MboII bound specifically to GAAGA sequences, producing two distinct complexes each containing one MboII and one DNA molecule. Interference analysis indicated that the initial species formed, named complex 1, comprised an interaction between the enzyme and the GAAGA target. Complex 2 involved interaction of the protein with both the GAAGA and the cutting sites. Only in the presence of divalent metal ions such as Ca2+ is the conversion of complex 1 to 2 rapid. Additionally, a very retarded complex was seen with Ca2+, possibly a (MboII)2-(DNA)2 complex. Plasmids containing a single GAAGA site were hydrolyzed slowly byMboII. Plasmids containing two sites were cut far more rapidly, suggesting that the enzyme requires two recognition sites in the same DNA molecule for efficient hydrolysis. MboII appears to have a mechanism similar to the best characterized type IIs enzyme, FokI. Both enzymes initially bind DNA as monomers, followed by dimerization to give an (enzyme)2-(DNA)2 complex. Dimerization is efficient only when the two target sites are located in the same DNA molecule and requires divalent metal ions.

The Escherichia coli vsr endonuclease recognises T:G base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5′ to the incorrectly paired T. The gene encoding the vsr endonuclease is next to the gene specifying the E. coli dcm DNA-methyltransferase; an enzyme that adds CH3 groups to the first dC within its target sequence CC[A/T]GG, giving C5MeC[A/T]GG. Deamination of the d5MeC results in CT[A/T]GG in which the first T is mis-paired with dG and it is believed that the endonuclease preferentially recognises T:G mismatches within the dcm recognition site. Here, the preference of the vsr endonuclease for bases surrounding the T:G mismatch has been evaluated. Determination of specificity constant (kst/KD; kst = rate constant for single turnover, KD = equilibrium dissociation constant) confirms vsr’s preference for a T:G mismatch within a dcm sequence i.e. CT[A/T]GG (the underlined T being mis-paired with dG) is the best substrate. However, the enzyme is capable of binding and hydrolysing sequences that differ from the dcm target site by a single base-pair (dcm star sites). Individual alteration of any of the four bases surrounding the mismatched T gives a substrate, albeit with reduced binding affinity and slowed turnover rates. The vsr endonuclease has a much lower selectivity for the dcm sequence than type II restriction endonucleases have for their target sites. The results are discussed in the light of the known crystal structure of the vsr protein and its possible physiological role.

Oligonucleotides labeled with hexachlorofluorescein (hex) have enabled the interaction of the restriction endonuclease EcoRV with DNA to be evaluated using fluorescence anisotropy. The sensitivity of hex allowed measurements at oligonucleotide concentrations as low as 1 nM, enabling K(D) values in the low nanomolar range to be measured. Both direct titration, i.e., addition of increasing amounts of the endonuclease to hex-labeled oligonucleotides, and displacement titration, i.e., addition of unlabeled oligonucleotide to preformed hex-oligonucleotide/EcoRV endonuclease complexes, have been used for K(D) determination. Displacement titration is the method of choice; artifacts due to any direct interaction of the enzyme with the dye are eliminated, and higher fluorescent-labeled oligonucleotide concentrations may be used, improving signal-to-noise ratio. Using this approach (with three different oligonucleotides) we found that the EcoRV restriction endonuclease showed a preference of between 1.5 and 6.5 for its GATATC target sequence at pH 7.5 and 100 mM NaCl, when the divalent cation Ca2+ is absent. As expected, both the presence of Ca2+ and a decrease in pH value stimulated the binding of specific sequences but had much less effect on nonspecific ones.

Colicin endonucleases and the H-N-H family of homing enzymes share a common active site structural motif that has similarities to the active sites of a variety of other nucleases such as the non-specific endonuclease from Serratia and the sequence-specific His-Cys box homing enzyme I-PpoI. In contrast to these latter enzymes, however, it remains unclear how H-N-H enzymes cleave nucleic acid substrates. Here, we show that the H-N-H enzyme from colicin E9 (the E9 DNase) shares many of the same basic enzymological characteristics as sequence-specific H-N-H enzymes including a dependence for high concentrations of Mg2+ or Ca2+ with double-stranded substrates, a high pH optimum (pH 8-9) and inhibition by monovalent cations. We also show that this seemingly non-specific enzyme preferentially nicks double-stranded DNA at thymine bases producing 3'-hydroxy and 5'-phosphate termini, and that the enzyme does not cleave small substrates, such as dinucleotides or nucleotide analogues, which has implications for its mode of inhibition in bacteria by immunity proteins. The E9 DNase will also bind single-stranded DNA above a certain length and in a sequence-independent manner, with transition metals such as Ni2+ optimal for cleavage but Mg2+ a poor cofactor. Ironically, the H-N-H motif of the E9 DNase although resembling the zinc binding site of a metalloenzyme does not support zinc-mediated hydrolysis of any DNA substrate. Finally, we demonstrate that the E9 DNase also degrades RNA in the absence of metal ions. In the context of current structural information, our data show that the H-N-H motif is an adaptable catalytic centre able to hydrolyse nucleic acid by different mechanisms depending on the substrate and metal ion regime.

Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the identification of small oligonucleotides that bind with high affinity and specificity to target proteins. Such DNAs/RNAs are a new class of potential chemotherapeutics that could block the enzymatic activity of pathologically relevant proteins. We have conducted a detailed biochemical study of the interaction of human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) with a SELEX-derived pseudoknot RNA aptamer. Electron paramagnetic resonance spectroscopy of site-directed spin-labeled RT mutants revealed that this aptamer was selected for binding to the "closed" conformation of the enzyme. Kinetic analysis showed that the RNA inhibitor bound to HIV RT in a two-step process, with association rates similar to those described for model DNA/DNA and DNA/RNA substrates. However, the dissociation of the pseudoknot RNA from RT was dramatically slower than observed for model substrates. Equilibrium binding studies revealed an extraordinarily low K(d), of about 25 pm, for the enzyme-aptamer interaction, presumably a consequence of the slow off-rates. Additionally, this pseudoknot aptamer is highly specific for HIV-1 RT, with the closely related HIV-2 enzyme showing a binding affinity close to 4 orders of magnitude lower.

Polymerases from the pol-I family which are able to efficiently use ddNTPs have demonstrated a much improved performance when used to sequence DNA. A number of mutations have been made to the gene coding for the Pol-II family DNA polymerase from the archaeon Pyrococcus furiosus with the aim of improving ddNTP utilisation, 'Rational' alterations to amino acids likely to be near the dNTP binding site (based on sequence homologies and structural information) did not yield the desired level of selectivity for ddNTPs. However, alteration at four positions (Q472, A486, L490 and Y497) gave rise to variants which incorporated ddNTPs better than the wild type, allowing sequencing reactions to be carried out at lowered ddNTP:dNTP ratios. Wild-type Pfu-Pol required a ddNTP:dNTP ratio of 30:1; values of 5:1 (Q472H), 1:3 (L490W), 1:5 (A486Y) and 5:1 (Y497A) were found with the four mutants; A486Y representing a 150-fold improvement over the wild type, A486, L490 and Y497 are on an alpha-helix that lines the dNTP binding groove, but the side chains of the three amino acids point away from this groove; Q472 is in a loop that connects this alpha-helix to a second long helix. None of the four amino acids can contact the dNTP directly, Therefore, the increased selectivity for ddNTPs is likely to arise from two factors: (i) small overall changes in conformation that subtly alter the nucleotide triphosphate binding site such that ddNTPs become favoured; (ii) interference with a conformational change that may be critical both for the polymerisation step and discrimination between different nucleotide triphosphates.