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G G Kneale

Publications and source records attributed to G G Kneale.

At least 19 recordsLinked to original sources
PubMed1996

Surface labelling of the type I methyltransferase M.EcoR124I reveals lysine residues critical for DNA binding.

The type IC methyltransferase M.EcoR124I consists of a specificity subunit (HsdS) and two methylation subunits (HsdM). Using chemical modifications, we have investigated the accessibility of lysine residues in the free enzyme and in the complex with its DNA recognition sequence. A total of 41 of the 109 lysine residues in the enzyme are susceptible to modification, of which 19 are located in the HsdS subunit and 11 in each of the two HsdM subunits. DNA binding results in extensive protection of lysine residues in the HsdS subunit, while those in the HsdM subunit are only protected weakly. The DNA binding activity of the methylase is abolished when a small fraction of the accessible lysine residues are modified. Peptide mapping and N-terminal sequencing has been used to locate the rapidly modified lysine residues in HsdS that are critical for DNA binding. Highly modified residues (K297, K261 and K327) are found in the C-terminal variable domain that is responsible for DNA recognition, but others (K196, K203 and K210) are found in the conserved regions that had not previously been implicated in DNA binding.

Amino Acid Sequence↗
PubMed1995

Probing the domain structure of the type IC DNA methyltransferase M.EcoR124I by limited proteolysis.

Limited proteolysis has been used to probe the domain structure of the type I DNA methyltransferase M.EcoR124I. Trypsin digestion of the methyltransferase generates two fragments derived from the HsdS subunit, a 28 kDa N-terminal domain and a 19 kDa C-terminal domain, leaving the HsdM subunit intact. Extensive digestion by chymotrypsin, however, removes 59 amino acid residues from the N terminus of the HsdM subunit to leave a 52 kDa C-terminal domain. Binding of the cofactor S-adenosyl methionine has no appreciable effect on the rate of cleavage, but binding of a 30 bp DNA duplex containing the cognate recognition sequence confers almost total protection. Following trypsin cleavage of the methyltransferase, a stable proteolytic product is produced which has been purified for biochemical characterisation. The trypsinised enzyme is shown to be a multimeric complex containing two intact HsdM subunits and both fragments of the HsdS subunit, consistent with the circular model proposed for the organisation of domains in the specificity subunit in type IC methyltransferases. Gel retardation studies show that the proteolysed enzyme still retains DNA binding activity, but its specificity for the DNA recognition sequence is dramatically reduced.

Amino Acid Sequence↗
PubMed1995

Comparison of Pf1 and Fd gene 5 proteins and their single-stranded DNA complexes by NMR spectroscopy and differential scanning calorimetry.

The Pf1 gene 5 protein forms a large helical nucleoprotein complex (Mr = 3.1 x 10(7)) with single-stranded viral DNA, from which a 32 amino acid sequence rich in alanine, proline, and glutamine residues can be removed from the C-terminus by limited proteolysis. Sharp resonances in the 1H NMR spectrum of the Pf1 nucleoprotein complex indicate that the C-terminal region of the protein subunits enjoys remarkable conformational flexibility in the complex. In contrast, the globular N-terminal domain of the protein subunits is rigidly held and does not contribute to the spectrum. The Fd gene 5 protein lacks this C-terminal flexible domain, and no distinct resonances can be observed in the 1H NMR spectrum when this protein is complexed to single-stranded viral DNA. Differential scanning calorimetry shows that the thermal stability of both the Pf1 and Fd gene 5 protein is increased by 8 degrees C in the complex with DNA, and the transition is highly cooperative. Removal of the C-terminal domain of the Pf1 gene 5 protein subunits has no appreciable effect either on the Tm of the DNA-protein complex or on the cooperative nature of the thermal transition. It is suggested that the C-terminal domain of the Pf1 gene 5 protein acts as a dynamic clamp which kinetically stabilizes the nucleoprotein complex.

Amino Acid Sequence↗
PubMed1995

Site-directed mutagenesis of the M13 gene 5 protein: the role of Arg-21, Tyr-26 and Tyr-41.

The gene 5 protein of bacteriophage M13 is a single stranded DNA binding protein essential for phage replication. We have generated the mutations R21A, Y26F and Y41A in the gene 5 protein and purified the mutant proteins for functional characterisation in vitro. The complex of Y26F with single-stranded DNA is disrupted at 0.8 M NaCl, the same salt concentration as that required to dissociate the native complex. However, the mutant proteins R21A and Y41A are considerably less stable and dissociate from single-stranded DNA at at 0.4 M NaCl. The fluorescence of the mutant proteins and the DNA-protein complexes they form has been compared with the wild-type protein to allow an assessment of the contribution from individual residues. We conclude that the fluorescence of Tyr-26 is 50% quenched in the complex with DNA, whereas that of Tyr-41 is fully quenched. Fluorescence titrations of the mutant proteins with poly(dT) show that all three mutant proteins can bind DNA but, in the case of Y41A, with a change of stoichiometry suggesting a loss of cooperativity. Gel retardation analysis of Y41A also shows anomalous behaviour in binding to oligonucleotides, consistent with the proposed involvement of Tyr-41 in dimer-dimer contacts in the nucleoprotein complex.

Arginine↗
PubMed1994

DNA-binding induces a major structural transition in a type I methyltransferase.

The type IC DNA methyltransferase M.EcoR124I is a complex multisubunit enzyme that recognizes the non-palindromic DNA sequence GAAN6RTCG. Small angle X-ray scattering has been used to investigate the solution structure of the methyltransferase and of complexes of the enzyme with unmethylated and hemimethylated 30 bp DNA duplexes containing the specific recognition sequence. A major change in the quaternary structure of the enzyme is observed following DNA binding, based on a decrease in the radius of gyration from 56 to 40 A and a reduction in the maximum dimension of the enzyme from 180 to 112 A. The structural transition observed is independent of the methylation state of the DNA. CD shows that there is no change in the secondary structure of the protein subunits when DNA is bound. In contrast, there is a large increase in the CD signal arising from the DNA, suggesting considerable structural distortion which may allow access to the bases targeted for methylation. We propose that DNA binding induces a large rotation of the two HsdM subunits towards the DNA, mediated by hinge bending domains in the specificity subunit HsdS.

Base Sequence↗
PubMed1994

A symmetrical model for the domain structure of type I DNA methyltransferases.

Type I DNA methyltransferases are complex multisubunit enzymes that methylate a specific base in each half of an asymmetric bipartite DNA recognition sequence. The specificity (S) subunit contains two corresponding DNA sequence recognition domains, plus a number of conserved regions which interact with two modification (M) subunits to form a trimeric enzyme of the form M2S. The way in which the subunits interact with DNA in a pseudo-symmetric fashion has long been unclear. Analysis of internal sequence repeats in the S-subunit shows the occurrence of significant homologies between the central conserved domain and sequences near the N and C termini. On the basis of this "split repeat", a "circular" organisation of the domains of this subunit is proposed that provides the required symmetry for interacting with the M-subunits and with the target DNA sequence. In the proposed model, one M-subunit interacts with the N and C-terminal conserved regions of the S-subunit, which are thereby brought into close proximity. The second M-subunit makes equivalent contacts with repeated sequences in the central conserved domain. The model suggests a more general scheme for the imposition of pseudo-dyad symmetry on protein subunits that have internal repeats by making equivalent contacts with additional subunits.

Amino Acid Sequence↗
PubMed1993

Cloning, expression and in vitro characterisation of the M13 gene 5 protein.

The gene 5 protein encoded in the genome of bacteriophage M13 is a single stranded DNA binding protein essential for phage replication. We have cloned a fragment of the M13 genome containing gene 5, and investigated the effect of upstream elements on expression of the gene by means of Bal 31 deletion analysis. The gene was also expressed from the lac promoter of the phagemid vector pUC119, and the recombinant protein purified and characterised for DNA binding. The affinity of the recombinant protein for single-stranded DNA was shown to be essentially identical to that of wild type gene 5 protein. Wild type gene 5 protein has a glutamic acid residue at position 30 which, on the basis of the crystal structure, was believed to play a role in maintaining the tertiary structure of the protein through the formation of a salt bridge with arginine-80. We show that substitution of glutamic acid at position 30 by lysine does not impair DNA binding, suggesting that a salt bridge between glutamate-30 and arginine-80 is not essential for the structural integrity of the gene 5 protein as previously proposed.

Amino Acid Sequence↗
PubMed1993

Tyr26 and Phe73 are essential for full biological activity of the Fd gene 5 protein.

Using site-saturation mutagenesis, we have established all possible amino acid substitutions at Tyr26 and Phe73 that are compatible with biological activity of the gene 5 protein in vivo. No substitutions were found at either site that gave rise to a fully functional gene 5 protein, indicating that these two amino acid residues are crucial. However, partial activity was found if either residue was replaced by another aromatic amino acid (Y26F, Y26W, F73Y, F73W). The results suggest that both Tyr26 and Phe73 are important for base stacking in the nucleoprotein complex. The functional consequences of the removal of the hydroxyl group from Tyr26 argue that this residue may, in addition, be involved in hydrogen bond formation to confer greater stability on the complex. In contrast, the addition of such a group to Phe73 reduces activity.

Amino Acid Sequence↗
PubMed1993

Identification of a compact DNA-binding domain in the gene 5 protein of Pf1 bacteriophage.

The structure of the gene 5 protein of filamentous bacteriophage Pf1 and its interaction with viral DNA have been investigated by a series of limited proteolysis experiments. The ability of purified proteolytic fragments of the Pf1 gene 5 protein to bind oligonucleotides and polynucleotides was monitored by gel retardation and fluorescence. The results show the presence of a compact DNA-binding "core" domain consisting of residues 1-112 of the protein, which is protected from proteolysis in the nucleoprotein complex. Digestion of the free gene 5 protein with subtilisin produces a smaller fragment (residues 7-102) which can no longer bind DNA. Although the N-terminal "core" domain shows full DNA binding activity by fluorescence, the gel retardation experiments suggest reduced kinetic stability of this domain in complexes with oligonucleotides, resulting from the removal of residues 113-144 from the C-terminus of the protein. The sequence of the C-terminal 32 amino acid residues is unusual, with a high proportion of alanine, glutamine, and proline residues which may be related to the role of this sequence in stabilizing the complex.

Amino Acid Sequence↗
PubMed1991

Structural parameters of the Pf1 gene 5 protein-DNA complex in solution by neutron scattering.

Neutron-scattering experiments have been performed on the intracellular complex formed by the gene 5 protein and single-stranded DNA in cells infected by filamentous bacteriophage Pf1. The contrast matched point of the complex (37% 2H2O) is lower than expected and implies that a substantial fraction of potentially labile hydrogen atoms are unable to exchange with the solvent. The mass/length ratio of the complex (3270 daltons/A) indicates an axial subunit repeat of 5.1 A, a value much larger than the subunit repeat previously determined in fibres. The measured value of the cross-sectional radius of gyration at infinite contrast (Rc = 43.3 A) indicates an outer radius of 60 to 63 A for the complex. The variation in Rc with contrast shows that regions of higher scattering density are located, on average, towards the outside of the complex. The high-angle region of the intensity curve (measured in 2H2O) reveals a clear subsidiary maximum at 0.105 A-1 arising from the 60 A helical pitch of the nucleoprotein complex. The structural parameters of the Pf1 gene 5 protein-DNA complex in solution are compared with those of the fd gene 5 protein-DNA complex.

Bacteriophages↗
PubMed1991

Circular dichroism and fluorescence analysis of the interaction of Pf1 gene 5 protein with poly(dT).

Circular dichroism (c.d.) and fluorescence spectroscopy have been used to investigate the interaction of the gene 5 protein of the filamentous bacteriophage Pf1 with single-stranded DNA. The c.d. spectrum of the Pf1 gene 5 protein is consistent with the absence of any significant alpha-helical content. The negative c.d. peak in the region of 210 nm, which arises from the protein, is diminished in the complex with poly(dT). Likewise, the c.d. peak at 265 nm arising from the poly(dT) decreases when the Pf1 gene 5 protein is bound, c.d. titrations of poly(dT) with Pf1 gene 5 protein indicate strong binding with a stoichiometry (n) of four nucleotides per protein subunit. In contrast, when the titrations were done using fluorescence anisotropy or fluorescence spectral shifts to follow binding, apparent stoichiometries between n = 2 and n = 4 were observed, often in the same experiment, depending on precise conditions. The results are interpreted in terms of two distinct modes of binding, in which either one or two subunits of the protein dimer are bound to the polynucleotide lattice, but still retaining the same local interaction with the DNA, with each binding site covering four nucleotides. The apparent stoichiometry of 2 results from the interaction of only one subunit of the dimer with the nucleic acid lattice, when protein is in excess. The second, unfilled, subunit of the dimer is nevertheless incorporated into the complex, resulting in the maximum possible fluorescence change when only half the sites are filled, since the fluorescence properties of the complex arise from protein-protein contacts associated with co-operative binding to the lattice. Further experiments in which the order of addition of components is changed, and the concentration of MgCl2 is varied, show that both of these factors are important in determining the dominant binding mode. In the absence of salt, dissociation and redistribution of the polynucleotide can occur following the addition of excess protein. This transition is suppressed in the presence of greater than 3 mM-MgCl2.

Bacteriophages↗
PubMed1991

The role of tyrosine residues in the DNA-binding site of the Pf1 gene 5 protein.

The 144 amino acid gene 5 protein of bacteriophage Pf1 binds tightly and cooperatively to single-stranded DNA during replication of the phage genome. It has been suggested that aromatic amino acid side chains are important for this interaction, probably through base stacking with the DNA. We have analysed the accessibility of tyrosine residues in the DNA-protein complex, and their importance to the DNA-binding activity of the protein, by chemical modification and protection experiments using tetranitromethane. Tyrosines 21, 30 and 55 are surface accessible in the free protein but are protected from modification in the complex with phage DNA. Moreover, modification of these residues in the free protein abolishes the ability to bind to DNA or oligonucleotides, as judged by fluorescence spectroscopy and gel retardation analysis. Modification of the protein also results in the formation of an intersubunit covalent cross-link between Tyr55 and Phe76, suggesting that Phe76 is located within the DNA-binding cleft of the protein. It is proposed that residues 17-34 of the Pf1 gene 5 protein form a beta-hairpin analogous to the 'DNA-binding wing' of the fd and Ike gene 5 proteins. We suggest the existence of a single-stranded DNA binding motif, in which Tyr30 of the Pf1 protein is equivalent to the functionally important Tyr26 of the fd gene 5 protein.

Amino Acid Sequence↗