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D P Hornby

Publications and source records attributed to D P Hornby.

At least 19 recordsLinked to original sources
PubMed2002

Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA methyltransferases.

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.

Antineoplastic Agents↗
PubMed2001

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity.

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2'-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with TAQ polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XBAI and by mass spectrometry of the PCR products.

Catalysis↗
PubMed2000

A positive selection vector combining tetracycline resistance that eliminates the need for bacterial plating.

We describe here the construction of a plasmid that combines positive selection with tetracycline resistance. The vector comprises a modified version of the gene encoding the cytosine-specific DNA methyltransferase MspI and a modified form of the pBR322 tetA(C) gene. The combination of these two genes facilitates the selection of recombinant plasmids in broth cultures, thereby eliminating the need for bacterial plating.

Bacteriological Techniques↗
PubMed1999

Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone.

DNA duplexes in which the target cytosine base is replaced by 2-H pyrimidinone have previously been shown to bind with a significantly greater affinity to C5-cytosine DNA methyltransferases than unmodified DNA. Here, it is shown that 2-H pyrimidinone, when incorporated into DNA duplexes containing the recognition sites for M.HgaI-2 and M.MspI, elicits the formation of inhibitory covalent nucleoprotein complexes. We have found that although covalent complexes are formed between 2-H pyrimidinone-modified DNA and both M.HgaI-2 and M.MspI, the kinetics of complex formation are quite distinct in each case. Moreover, the formation of a covalent complex is still observed between 2-H pyrimidinone DNA and M.MspI in which the active-site cysteine residue is replaced by serine or threonine. Covalent complex formation between M.MspI and 2-H pyrimidinone occurs as a direct result of nucleophilic attack by the residue at the catalytic position, which is enhanced by the absence of the 4-amino function in the base. The substitution of the catalytic cysteine residue by tyrosine or chemical modification of the wild-type enzyme with N-ethylmaleimide, abolishes covalent interaction. Nevertheless the 2-H pyrimidinone-substituted duplex still binds to M.MspI with a greater affinity than a standard cognate duplex, since the 2-H pyrimidinone base is mis-paired with guanine.

Binding Sites↗
PubMed1998

Tyrosine phosphorylation of a 95 kDa protein and induction of the acrosome reaction in human spermatozoa by recombinant human zona pellucida glycoprotein 3.

Protein tyrosine phosphorylation and induction of the acrosome reaction (AR) in non-capacitated and capacitated human spermatozoa was investigated in response to recombinant human zona pellucida glycoprotein (rhZP3) produced by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA. rhZP3-containing medium promoted the AR in a high proportion of capacitated spermatozoa (48.6 +/- 3.2%; P < 0.01) compared with control (no rhZP3) samples (14.8 +/- 2.1%). However, rhZP3-containing medium did not cause increased acrosomal exocytosis in non-capacitated spermatozoa (16.8 +/- 3.0%). Induction of the AR was associated with increased tyrosine phosphorylation of a 95 +/- 5 kDa epitope only in capacitated spermatozoa. A dose-dependent increase in the protein phosphorylation of a 95 kDa epitope in response to rhZP3 was detected by [gamma-32P]-ATP labelling of detergent-solubilized sperm proteins. When spermatozoa were co-incubated with monoclonal antibody 97.25 (mAb 97.25) recognizing a 95 kDa tyrosine kinase epitope, there was no rhZP3 induction of tyrosine phosphorylation of the 95 kDa protein. Such co-incubation also markedly inhibited the AR (23.9 +/- 3.1%). These results support the model that initial interaction of the fertilizing spermatozoon with ZP3 involves the tyrosine phosphorylation of a 95 kDa tyrosine kinase protein and that this requires capacitation.

Acrosome Reaction↗
PubMed1996

Activation of a yeast pseudo DNA methyltransferase by deletion of a single amino acid.

The biological methylation cytosine bases in DNA is central to such diverse phenomena as restriction and modification in bacteria, repeat induced point-mutation (RIPing) in fungi and for programming gene expression patterns in vertebrates. Structural studies on HhaI DNA methyltransferase, together with the sequence comparisons of around 40 cytosine-specific DNA methyltransferases, have recently provided a molecular framework for understanding the mechanism of action of the related group of enzymes that catalyse this base modification. There are, however, a number of organisms, including Saccharomyces cerevisiae, Schizosaccharomyces pombe and Drosophila melanogaster, which have no detectable DNA methylation. Here we report that the product of the pmt1 gene recently identified in S. pombe, which contains most of the primary structure elements of a typical cytosine-specific DNA methyltransferase, is catalytically inert owing to the insertion of a Ser residue between the Pro-Cys motif found at the active site of all such DNA methyltransferases. Following deletion of this Ser residue, catalytic activity is restored and, using a range of DNA binding experiments, it is shown that the enzyme recognises and methylates the sequence CC(A/T)GG, the same sequence that is modified by the product of the Escherichia coli dcm gene. The pmt gene of S. pombe therefore encodes a pseudo DNA methyltranferase, which we have called psiM.SpoI.

Base Sequence↗
PubMed1996

Biological activity of recombinant human ZP3 produced in vitro: potential for a sperm function test.

The human zona binding test is the most predictive test of sperm function yet the availability of human zona severely restricts its clinical use. The primary aim of this study was to use a commercially available in-vitro transcription and translation system to produce immobilized recombinant human ZP3 (rhuZP3) on agarose beads. The biological activity of this preparation was examined using sperm binding and the acrosome reaction. Significantly higher levels of sperm binding to rhuZP3 beads (n = 12, P < 0.05) compared with controls were observed and there was a significant induction (n = 12, P < 0.01) in the acrosome reaction after overnight incubation at 37 degrees C in 5% CO2 in air. In conclusion, the in-vitro transcription and translation system can produce sufficient quantities of purified immobilized biologically active rhuZP3. These preliminary experiments will enable further refinements to be made so that a solid-phase sperm function test based on rhuZP3 coated beads is likely to be developed in the near future.

Acrosome↗
PubMed1996

Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa.

Recombinant human ZP3 (rhuZP3) generated by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA was used to study the acrosome reaction (AR) and intracellular calcium fluxes in capacitated human spermatozoa. Conditioned medium containing rhuZP3 significantly induced the AR (P < or = 0.005) in 59.4 +/- 4.7% of spermatozoa (control = 8.5 +/- 3.1%) and caused complete acrosomal loss in a further 17.2 +/- 3.8% of cells (control = 3.7 +/- 0.7%; mean +/- SEM, n = 5). Sperm motility was not affected and acrosomal exocytosis in response to rhuZP3 was also shown to be time-dependent. Basal concentrations of sperm intracellular calcium were measured (82 +/- 7 nM; mean +/- SEM, n = 9). A transient increase in intracellular calcium (typically up to 400-450 nM) occurred within 1 min of rhuZP3 addition and was followed by sustained lower values of calcium (200-400 nM). These responses were dependent on the amount of rhuZP3. This is the first report of zona protein-induced changes in intracellular calcium levels in human spermatozoa. The results support the premise that ZP3 is an agonist of the human sperm AR and that rhuZP3 generated in a eukaryotic cell is effective in this respect.

Acrosome↗
PubMed1995

Identification of direct-repeat-binding protein 1 (DRP-1), a DNA-binding protein that binds specifically to the 'malic' enzyme gene promoter direct repeat element.

The 'malic' enzyme (ME) gene promoter contains three main regulatory regions. One of these, the direct repeat element (DRE), contains tandem degenerate Sp1-binding sites separated by a 3 bp intervening sequence. We now show that a previously unreported 95 kDa protein, which we have designated DRP-1, binds strongly to the DRE region in a highly specific manner. Western-blot analysis confirms that this protein is not Sp1, which has been shown to bind to similar degenerate sites. Competitive binding assays using purified DRP-1 further reveal that neither non-specific nor Sp1-consensus-site-containing oligonucleotides can displace those complexes formed between DRP-1 and the DRE sequence, thus confirming sequence-specific binding by this protein. SDS/PAGE analysis of DRE-protein complexes isolated by direct excision and transplantation from retardation gels confirms the presence of the 95 kDa protein and, in addition, suggests that more than one binding site exists for this protein within the DRE. This is in accord with the repeated nature of the DRE DNA sequence which contains two CACC box motifs.

Base Sequence↗
PubMed1994

The DNA recognition subunit of a DNA methyltransferase is predominantly a molten globule in the absence of DNA.

Enzyme-catalysed DNA methylation provides an opportunity for the modulation of protein-DNA recognition in biological systems. Recently we have demonstrated that the smaller of the two subunits of the heterodimeric, cytosine-specific DNA methyltransferase, M. AquI, is largely responsible for sequence-specific DNA recognition. Here we present evidence from a series of NMR, fluorescence and circular dichroism spectroscopy experiments that the DNA binding subunit of M. AquI has the characteristics of a molten globule in the absence of the catalytic machinery. In this metastable state this subunit retains its ability to bind DNA in a sequence-specific manner. We believe this finding offers an insight into the structural flexibility which underpins the mechanism of action of these enzymes, and may provide a possible biological role for molten globules in protein function.

Amino Acid Sequence↗
PubMed1994

Site and significance of chemically modifiable cysteine residues in glutamate dehydrogenase of Clostridium symbiosum and the use of protection studies to measure coenzyme binding.

Protein chemical studies of NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.2) from Clostridium symbiosum indicate only two cysteine residues/subunit, in good agreement with the gene sequence. Experiments with various thiol-modifying reagents reveal that in native clostridial GDH only one of these two cysteines is accessible for reaction. This residue does not react with iodoacetate, iodoacetamide, N-ethylmaleimide or N-phenylmaleimide, but reaction with either p-chloromercuribenzene sulphonate or 5,5'-dithiobis(2-nitrobenzoic acid) causes complete inactivation, preventable by NAD+ or NADH but not by glutamate or 2-oxoglutarate. Protection studies with combinations of substrates show that glutamate enhances protection by NADH, whereas 2-oxoglutarate diminishes it. These studies were also used to determine a dissociation constant (0.69 mM) for the enzyme-NAD+ complex. Similar data for NADH indicated mildly cooperative binding with a Hill coefficient of 1.32. The significance of these results is discussed in the light of the high-resolution crystallographic structure for clostridial GDH and in relation to information for GDH from other sources.

Amino Acid Sequence↗
PubMed1994

Ligand-induced conformational states of the cytosine-specific DNA methyltransferase M.HgaI-2.

The interaction of one of the two DNA methyltransferases encoded by the HgaI restriction and modification system, M.HgaI-2, with substrates and substrate analogues is described. Circular dichroism spectroscopy has been used to demonstrate that addition of the methyl donor, S-adenosyl-L-methionine and the inhibitory substrate analogue sinefungin, both induce conformational transitions in the protein in the absence of DNA. Moreover, the addition of DNA is shown to enhance the apparent secondary structure of M.HgaI-2 whilst addition of sinefungin or S-adenosyl-L-methionine reduces apparent secondary structure. The circular dichroism spectrum of the abortive complex between the enzyme, DNA and sinefungin is dominated by the conformational properties of the binary complex of enzyme and sinefungin alone. Addition of a specific oligodeoxynucleotide duplex in which the target cytosine is replaced by a pyrimidinone, leads to a further ligand induced conformational transition as determined by electrophoretic analysis. The addition of sinefungin, or S-adenosyl-L-methionine, to M.HgaI-2 bound to the reactive oligodeoxynucleotide duplex, leads to yet another conformational transition in the protein as determined by the differential susceptibility of ternary and binary complexes to proteolysis. These experiments identify at least six ligand-inducible conformational states of M.HgaI-2 and, in view of the sequence similarity amongst this class of enzymes, suggest that conformational flexibility is a general feature of C-5 cytosine-specific DNA methyltransferases. Moreover, the substitution of the target cytosine by a pyrimidinone mimics the effect of 5-azacytosine incorporation into DNA.

Adenosine↗
PubMed1993

Determination of the order of substrate addition to MspI DNA methyltransferase using a novel mechanism-based inhibitor.

The cloning and overexpression of the MspI DNA methyltransferase as a functional fusion with glutathione S-transferase is described. The fusion enzyme retains full biological activity and has been used to investigate the interaction of substrates and inhibitors with MspI DNA methyltransferase. The fusion enzyme has been purified to homogeneity in a single step on GSH-agarose and is free from contaminating exonuclease activity. The enzyme can be photolabelled with S-adenosyl-L-methionine and the level of incorporation of label is enhanced by the presence of a nonspecific DNA duplex. In the presence of a cognate oligodeoxynucleotide, no photolabelling was observed since methyl transfer occurs instead. The inclusion of a mechanism-based inhibitor of C-5 deoxycytidine DNA methylation (an oligodeoxynucleotide containing the base 2-pyrimidinone-1-beta-D-2'-deoxyribofuranoside in the position of the deoxycytidine to which methyl addition occurs), which is thought to form a covalent interaction with the reactive cysteine of such enzymes, led to an enhancement of S-adenosyl-L-methionine photolabelling which suggests that, in contrast with results obtained with EcoRII DNA methyltransferase [Som and Friedman (1991) J. Biol. Chem. 266, 2937-2945], methylcysteine is not the photolabelled product. The implications of the results obtained with this mechanism-based inhibitor are discussed with respect to other C-5-specific DNA methyltransferases. Gel-retardation assays in the presence of cognate oligodeoxynucleotides that contain the reactive pyrimidinone base in place of the deoxycytidine target base are described. These demonstrate that most probably a stable covalent bond is formed between the methyltransferase and this oligodeoxynucleotide. However, the alternative of extremely tight non-covalent binding cannot be rigorously excluded. Furthermore, the results from these experiments indicate that the reaction mechanism proceeds in a manner similar to that of HhaI DNA methyltransferase with sequence-specific DNA binding being followed by addition of S-adenosyl-L-methionine and concomitant isomerization of the ternary complex leading to methyl transfer. S-Adenosyl-L-homocysteine appears to inhibit the reaction pathway as a result of either competition with the methyl donor and potentiation of a high-affinity interaction between the enzyme and DNA in an abortive ternary complex or through an allosteric interaction.

Base Sequence↗
PubMed1993

Effects of co-factor and deoxycytidine substituted oligonucleotides upon sequence-specific interactions between MspI DNA methyltransferase and DNA.

MspI methyltransferase (M.MspI) catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C-5 position of the outer deoxycytidine base in the DNA sequence 5'-CCGG-3'. Recombinant M.MspI when expressed and purified as a translational fusion with glutathione-S-transferase, shows all of the properties of the wild-type enzyme. We report the kinetic analysis of M.MspI binding to DNA, which suggests a two-stage methylation process, whose initial DNA binding rate is governed by the presence of a positively charged sulphonium centre on the cofactor. Results are also presented that indicate that M.MspI binds preferentially to hemi-methylated DNA and that full methylation of either deoxycytidine on both strands significantly impairs sequence-specific protein-DNA interactions. Furthermore, the importance of the 4-amino group of the inner deoxycytidine for sequence-specific protein-DNA interactions is demonstrated by substituting deoxycytidine with 2-pyrimidinone-1-beta-D-2-deoxyriboside. In addition, we detail the intrinsic structural elements of a cofactor, required to enhance the binding of M.MspI to its recognition sequence, by using S-adenosyl-L-methionine and a range of derivatives.

Base Sequence↗
PubMed1993

Modulation of human DNA methyltransferase activity and mRNA levels in the monoblast cell line U937 induced to differentiate with dibutyryl cyclic AMP and phorbol ester.

The regulation of DNA (cytosine-5) methyltransferase (DNA MeTase) enzyme activity and gene expression was examined in the monoblastoid U937 cell line induced to differentiate with either dibutyryl cyclic AMP (dbcAMP) or phorbol ester. dbcAMP treatment was found to cause the rapid (< 4 h) suppression of DNA MeTase specific activity, with no DNA MeTase activity detectable after 10 h. Equally, no DNA MeTase activity was detectable in nuclear extracts of fresh peripheral blood monocytes. Using both a U937 DNA MeTase cDNA and a mouse DNA MeTase cDNA as probes, steady-state levels of DNA MeTase mRNA were found to decline sharply between 4 and 15 h after dbcAMP treatment. No DNA MeTase mRNA was detectable after 20 h of dbcAMP treatment. Nuclear run-on analysis showed there to be only a small (40%) suppression of DNA MeTase gene transcription in cells treated with dbcAMP for 24 h, implying a role for post-transcriptional processes in the regulation of DNA MeTase mRNA levels. The observed decline in DNA MeTase activity/mRNA levels appeared to precede the dbcAMP-induced arrest in DNA replication, as judged by the incorporation of tritiated thymidine into DNA. In contrast to the effect of dbcAMP, treatment of U937 cells with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) led to an overall stimulation of DNA MeTase specific activity. The TPA response was found to be complex and broadly consisted of an early (0-15 h) burst of DNA MeTase activity followed by a more gradual sustained increase in DNA MeTase activity after prolonged (16-40 h) TPA treatment. The early phase of high DNA MeTase activity was not mirrored by an increase in steady-state levels of DNA MeTase mRNA, as judged by Northern blot analysis. However, a substantial induction of DNA MeTase mRNA levels was observed after 20-24 h of TPA treatment. Nuclear run-on analysis showed this not to be due to any significant increase in DNA MeTase gene transcription. The observed increases in DNA MeTase activity/mRNA levels were observed whilst cells were undergoing deproliferation. Interestingly, the addition of TPA and more physiological protein kinase C (PKC) activators, such as diacylglycerol and phosphatidylserine, to DNA MeTase-enriched nuclear extracts generated a 4.5-fold and a 1.5-fold increase in DNA MeTase specific activity respectively. The TPA-induced stimulation of DNA MeTase activity could be inhibited by the PKC inhibitor H-9, implicating a role for PKC in the regulation of DNA MeTase activity in vivo.

Bucladesine↗