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L M Powell

Publications and source records attributed to L M Powell.

At least 19 recordsLinked to original sources
PubMed2001

Target recognition by EcoKI: the recognition domain is robust and restriction-deficiency commonly results from the proteolytic control of enzyme activity.

We report a genetic and biochemical analysis of a target recognition domain (TRD) of EcoKI, a type I restriction and modification enzyme. The TRDs of type I R-M systems are within the specificity subunit (HsdS) and HsdS confers sequence specificity to a complex endowed with both restriction and modification activities. Random mutagenesis has revealed that most substitutions within the amino TRD of EcoKI, a region comprising 157 amino acid residues, have no detectable effect on the phenotype of the bacterium, even when the substitutions are non- conservative. The structure of the TRD appears to be robust. All but one of the six substitutions that confer a restriction-deficient, modification-deficient (r(-)m(-)) phenotype were found to be in the interval between residues 80 and 110, a region predicted by sequence comparisons to form part of the protein-DNA interface. Additional site-directed mutations affecting this interval commonly impair both restriction and modification. However, we show that an r(-) phenotype cannot be taken as evidence that the EcoKI complex lacks endonuclease activity; in response to even a slightly impaired modification efficiency, the endonuclease activity of EcoKI is destroyed by a process dependent upon the ClpXP protease. Enzymes from mutants with an r(-)m(-) phenotype commonly retain some sequence-specific activity; methylase activity can be detected on hemimethylated DNA substrates and residual endonuclease activity is implied whenever the viability of the r(-)m(-) bacterium is dependent on ClpXP. Conversely, the viability of ClpX(-) r(-)m(-) bacteria can be used as evidence for little, or no, endonuclease activity. Of 14 mutants with an r(-)m(-) phenotype, only six are viable in the absence of ClpXP. The significance of four of the six residues (G91, G105, F107 and G141) is enhanced by the finding that even conservative substitutions for these residues impair modification, thereby conferring an r(-)m(-) phenotype.

Adenosine Triphosphatases↗
PubMed1998

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.

Base Sequence↗
PubMed1998

Sequence-specific DNA binding by EcoKI, a type IA DNA restriction enzyme.

The type I DNA restriction and modification enzymes of prokaryotes are multimeric enzymes that cleave unmethylated, foreign DNA in a complex process involving recognition of the methylation status of a DNA target sequence, extensive translocation of DNA in both directions towards the enzyme bound at the target sequence, ATP hydrolysis, which is believed to drive the translocation possibly via a helicase mechanism, and eventual endonucleolytic cleavage of the DNA. We have examined the DNA binding affinity and exonuclease III footprint of the EcoKI type IA restriction enzyme on oligonucleotide duplexes that either contain or lack the target sequence. The influence of the cofactors, S-adenosyl methionine and ATP, on binding to DNA of different methylation states has been assessed. EcoKI in the absence of ATP, with or without S-adenosyl methionine, binds tightly even to DNA lacking the target site and the exonuclease footprint is large, approximately 45 base-pairs. The protection is weaker on DNA lacking the target site. Partially assembled EcoKI lacking one or both of the subunits essential for DNA cleavage, is unable to bind tightly to DNA lacking the target site but can bind tightly to the recognition site. The addition of ATP to EcoKI, in the presence of AdoMet, allows tight binding only to the target site and the footprint shrinks to 30 base-pairs, almost identical to that of the modification enzyme which makes up the core of EcoKI. The same effect occurs when S-adenosyl homocysteine or sinefungin are substituted for S-adenosyl methionine, and ADP or ATPgammaS are substituted for ATP. It is proposed that the DNA binding surface of EcoKI comprises three regions: a "core" region which recognises the target sequence and which is present on the modification enzyme, and a region on each DNA cleavage subunit. The cleavage subunits make tight contacts to any DNA molecule in the absence of cofactors, but this contact is weakened in the presence of cofactors to allow the protein conformational changes required for DNA translocation when a target site is recognised by the core modification enzyme. This weakening of the interaction between the DNA cleavage subunits and the DNA could allow more access of exonuclease III to the DNA and account for the shorter footprint.

Adenosine Triphosphate↗
PubMed1998

EcoKI with an amino acid substitution in any one of seven DEAD-box motifs has impaired ATPase and endonuclease activities.

For type I restriction systems, recently determined nucleotide sequences predict conserved amino acids in the subunit that is essential for restriction but not modification (HsdR). The conserved sequences emphasize motifs characteristic of the DEAD-box family of proteins which comprises putative helicases, and they identify a new candidate for motif IV. We provide evidence based on an analysis of Eco KI which supports both the relevance of DEAD-box motifs to the mechanism of restriction and the new definition of motif IV. Amino acid substitutions within the newly identified motif IV and those in six other previously identified DEAD-box motifs, but not in the original motif IV, confer restriction-deficient phenotypes. We have examined the relevance of the DEAD-box motifs to the restriction pathway by determining the steps permitted in vitro by the defective enzymes resulting from amino acid substitutions in each of the seven motifs. Eco KI purified from the seven restriction-deficient mutants binds to an unmethylated target sequence and, in the presence of AdoMet, responds to ATP by undergoing the conformational change essential for the pathway of events leading to DNA cleavage. The seven enzymes have little or no ATPase activity and no endonuclease activity, but they retain the ability to nick unmodified DNA, though at reduced rates. Nicking of a DNA strand could therefore be an essential early step in the restriction pathway, facilitating the ATP-dependent translocation of DNA, particularly if this involves DNA helicase activity.

Adenosine Triphosphatases↗
PubMed1995

Tyrosine 27 of the specificity polypeptide of EcoKI can be UV crosslinked to a bromodeoxyuridine-substituted DNA target sequence.

The specificity (S) subunit of the restriction enzyme EcoKI imparts specificity for the sequence AAC(N6)GTGC. Substitution of thymine with bromodeoxyuridine in a 25 bp DNA duplex containing this sequence stimulated UV light-induced covalent crosslinking to the S subunit. Crosslinking occurred only at the residue complementary to the first adenine in the AAC sequence, demonstrating a close contact between the major groove at this sequence and the S subunit. Peptide sequencing of a proteolytically-digested, crosslinked complex identified tyrosine 27 in the S subunit as the site of crosslinking. This is consistent with the role of the N-terminal domain of the S subunit in recognizing the AAC sequence. Tyrosine 27 is conserved in the S subunits of the three type I enzymes that share the sequence AA in the trinucleotide component of their target sequence. This suggests that tyrosine 27 may make a similar DNA contact in these other enzymes.

Amino Acid Sequence↗
PubMed1995

S-adenosyl methionine alters the DNA contacts of the EcoKI methyltransferase.

The EcoKI methyltransferase methylates two adenines on opposite strands of its bipartite DNA recognition sequence AAC(N6)GTGC. The enzyme has a strong preference for hemimethylated DNA substrates, but the methylation state of the DNA does not influence its binding affinity. Methylation interference was used to compare the contacts made by the EcoKI methyltransferase with unmodified, hemimethylated or fully modified DNAs. Contacts were seen at or near the N7 position of guanine, in the major groove, for all of the guanines in the EcoKI recognition sequence, and at two guanines on the edge of the intervening spacer sequence. The presence of the cofactor and methyl donor S-adenosyl methionine had a striking effect on the interference pattern for unmodified DNA which could not be mimicked by the presence of the cofactor analogue S-adenosyl homocysteine. In contrast, S-adenosyl methionine had no effect on the interference patterns for either kind of hemimethylated DNA, or for fully modified DNA. Differences between the interference patterns for the unmodified DNA and any of the three forms of methylated DNA provide evidence that methylation of the target sequence influences the conformation of the protein-DNA interface, and illustrate the importance of S-adenosyl methionine in the distinction between unmodified and methylated DNA by the methyltransferase.

Base Sequence↗
PubMed1993

DNA recognition by the EcoK methyltransferase. The influence of DNA methylation and the cofactor S-adenosyl-L-methionine.

The methyltransferase of the EcoK type I restriction/modification system is trimeric, M2S1, where the S subunit determines the sequence specificity of the enzyme. The methyltransferase has a strong preference for hemimethylated substrate DNA and, therefore, we have investigated the effect of the methylation state of DNA on binding by the enzyme, together with the effects on binding of the cofactor S-adenosyl-L-methionine. Our results indicate that the methyltransferase has two non-interacting S-adenosyl-L-methionine binding sites, each with a dissociation constant of 3.60 (+/- 0.42) microM determined by equilibrium dialysis, or 2.21 (+/- 0.29) microM determined by the displacement of a fluorescent probe. Ultraviolet light-induced crosslinking showed that S-adenosyl-L-methionine binds strongly only to the modification (M) subunits. Changes in the sedimentation velocity of the methyltransferase imply a protein conformational change due to S-adenosyl-L-methionine binding. Gel retardation results show that the binding of S-adenosyl-L-methionine to the methyltransferase enhances binding to both specific and non-specific DNAs, but the enhancement is greater for the specific DNA. Differences in binding affinities contribute to the recognition of the specific nucleotide sequence AAC(N)6GTGC by the methyltransferase in preference to a non-specific sequence. In contrast, although the complexes of unmodified and hemimethylated DNAs with the methyltransferase have different mobilities in non-denaturing gels, there appears to be no contribution of binding affinity to the distinction between these two substrates. Therefore, the preference for a hemimethylated substrate must be due to a difference in catalysis.

Allosteric Regulation↗
PubMed1993

Immune-mediated skin disease in the dog and cat.

Immune-mediated skin disease was recorded in 24 dogs and 11 cats by the Department of Pathology and Microbiology, University of Bristol, over the period 1973-1992. The clinical and histopathological features, together with the age, breed and sex, are given for each case. Skin biopsies from the 24 dogs in this series, together with tissue from 25 control dogs, were immunostained for IgG, IgM and C3 by direct immunoperoxidase methodology. The same technique, with species-specific antisera for IgG and IgM, was applied to skin biopsies from 10 affected cats. The correlation between clinical features, histopathology and immunohistochemistry is discussed.

Animals↗
PubMed1992

Effects of glycosylation on the folding and stability of human, recombinant and cleaved alpha 1-antitrypsin.

The equilibrium unfolding transitions for the human M form of alpha 1-antitrypsin have been determined using a number of techniques reflecting changes in tryptophan fluorescence lifetime and quenching, exposure of tryptophan to solvent, secondary structure and the Stokes' radius of the protein. The denaturation curves are more complex than is usual for globular proteins and indicate the presence of multiple equilibrium intermediates in the presence of denaturant. This is in marked contrast to the more co-operative transition of the cleaved inhibitor. In addition, a recombinant non-glycosylated alpha 1-antitrypsin has been shown to have a closely similar conformation to the human M protein and to exhibit very similar reversible unfolding transitions, and hence similar stability and co-operativity. Differences in tryptophan environment are reflected in the dequenching of tryptophan fluorescence and reduced asymmetry in the near ultraviolet circular dichroism of the non-glycosylated protein, suggesting direct interaction of glycosyl residues with a tryptophan. Both the M type and the recombinant protein exhibit similar patterns of folding, with rapid collapse to a compact intermediate reminiscent of the widely observed molten globule state that folds more slowly to the native protein. The papain-cleaved M form also folds through a similar compact state in the absence of the C-terminal peptide that results from cleavage. It is concluded that part of the C-terminal 36 residue peptide interacts strongly with the main body of the protein in the folded inhibitor. This interaction will also be important during early stages of folding of the intact protein to direct the folding pathway. The lack of glycosylation leads to an increase in aggregation of the recombinant protein upon refolding, especially after extended denaturation times. The more rapid turnover of the recombinant protein in vivo is shown not to be due to a lower thermodynamic stability, but may be associated with a lower kinetic stability arising from the increased tendency to aggregation.

Chromatography, Gel↗
PubMed1991

An additional editing site is present in apolipoprotein B mRNA.

Human intestinal apolipoprotein (apo) B mRNA undergoes a C to U RNA editing at nucleotide 6666 to generate a translation stop at codon 2153, which defines the carboxy-terminal of apo B48. Here we show that two of eleven human intestinal cDNAs spanning residue 6666 were edited from a genomically-encoded C to a T at residue 6802 as well as at residue 6666. This additional editing converts Thr (ACA) codon 2198 to Ile (AUA). Synthetic RNA including the nucleotide 6802 was edited in vitro by intestinal extracts at 10-15% of the editing efficiency of nucleotide 6666. A sequence is identified as important for recognition by the editing activity. No secondary structural homology was identified between the two edited sites. No other sequence in the region between 6411 and 6893 nucleotides of apo B mRNA was found to be edited in vivo or in vitro. Apo B RNA editing extracts from intestine did not edit maize cytochrome oxidase II mRNA.

Apolipoproteins B↗
PubMed1991

Interaction of recombinant apolipoprotein(a) and lipoprotein(a) with macrophages.

Elevated plasma levels of lipoprotein(a), Lp(a), represent a major, inherited risk factor for coronary heart disease, although the mechanism of its action remains unknown. Lp(a) is distinguished from the related LDL particle by the addition of apolipoprotein(a), apo(a). The presence of this large glycoprotein is likely to affect the binding of the particle to the LDL receptor and/or other receptors which may contribute to the atherogenic potential of Lp(a). Here we demonstrate the binding to macrophages of Lp(a) and pure recombinant apo(a) protein, via a specific, high-affinity receptor. This binding could lead to foam cell formation and the localization of Lp(a) to atherosclerotic plaques.

Animals↗
PubMed1989

Sequence requirements for apolipoprotein B RNA editing in transfected rat hepatoma cells.

Apolipoprotein (apo) B mRNA undergoes a novel tissue-specific editing reaction, which replaces a genomically templated cytidine with uridine. This substitution converts codon 2153 from glutamine (CAA) in apo B100 mRNA to a stop codon (UAA) in apoB48 mRNA (Powell, L. M., Wallis, S. C., Pease, R. J., Edwards, Y. H., Knott, T. J., and Scott, J. (1987) Cell 50, 831-840). To examine sequences in the human apoB mRNA required for the editing reaction, a series of deletion mutants around the cytidine conversion site was prepared and transfected into a rat hepatoma cell line (McArdle 7777). This cell makes both apoB100 and apoB48. Editing was detected by a primer extension assay on cDNA that had been amplified by the polymerase chain reaction. RNAs of between 2385 and 26 nucleotides spanning the conversion site underwent similar levels of conversion. Editing was confirmed by cloning and sequencing of cDNA corresponding to the transfected RNAs. Conversion did not occur in transfected human hepatoblastoma (HepG2) or epithelial carcinoma (HeLa) cell lines, which do not make apoB48. These results verify that apoB48 is generated by a genuine tissue-specific RNA editing reaction and show that 26 nucleotides of apoB mRNA are sufficient for editing.

Animals↗
PubMed1989

RNA editing: a novel mechanism for regulating lipid transport from the intestine.

Human apolipoprotein (apo)-B mRNA undergoes a novel tissue specific editing reaction which replaces a genomically templated cytidine with uridine. This substitution converts codon 2153 from glutamine (CAA) in apo-B100 mRNA to a stop codon (UAA) in apo-B48 mRNA. This novel RNA editing process is responsible for the generation of hepatic apo-B100 and intestinal apo-B48. We have established the following concerning this process: (1) by transfection of a series of deletion mutants into the rat hepatoma cell line McArdle 7777, which makes both apo-B100 and apo-B48, we have defined a minimum sequence of 26 nucleotides that is required for apo-B mRNA editing. The sequence containing the modified nucleotide forms a 26 nucleotide highly conserved stem loop with the modified nucleotide occurring in an 8-base loop. (2) Conversion in vitro of apo-B mRNA has been established, using cell free S100 cytoplasmic extract and synthetic RNA templates. Activity was abolished by protease treatment. (3) Transgenic mice were created which expressed a human apo-B construct spanning the stop codon. Apo-B mRNA was found in all tissues examined and this was shown to undergo editing. (4) In the rat liver, which produces apo B-100 and apo-B48, modulation of the relative proportion of these proteins by thyroxine was demonstrated to be mediated at the level of the RNA editing mechanism. It is concluded that apo-B mRNA is edited by a generally expressed protein and editing is highly regulated.

Animals↗
PubMed1988

Thyroid hormone modulates the introduction of a stop codon in rat liver apolipoprotein B messenger RNA.

Apolipoprotein B (apoB) biosynthesis by rat liver was studied following thyroid hormone (3,5,3'-triiodo-L-thyronine) administration to hypothyroid rats. Pharmacologic doses of 3,5,3'-triiodo-L-thyronine caused suppression of apoB100 synthesis but did not affect apoB48 levels. There was no detectable apoB100 synthesis in hyperthyroid rats. To examine whether these results were mediated by the previously demonstrated mechanism of RNA modification (Powell, L. M., Wallis, S. C., Pease, R. J., Edwards, Y. H., Knott, T. J., and Scott, J. (1987) Cell 50, 831-840), the DNA sequence corresponding to the C-terminal end of rat apoB48 was determined from rat liver cDNA clones. Rat cDNAs contained a stop codon at an identical position to that found in human and rabbit apoB48 intestinal cDNA. To quantitate the relative amounts of apoB100 and apoB48 message, cDNA was synthesized from hepatic and intestinal apoB RNA and a 207-base pair fragment amplified using the polymerase chain reaction. The products were then differentially hybridized with oligonucleotides specific for apoB100 (containing CAA) or apoB48 (TAA). Control and hypothyroid liver contained approximately equal amounts of CAA and TAA, while hyperthyroid liver contained greater than 90% TAA. All gut samples contained 94-98% TAA. Genomic DNA from rat liver contained only CAA. The results demonstrate that apoB mRNA modification can be hormonally modulated in the adult rat by induction of a mechanism involving substitution of a stop codon into hepatic apoB100 mRNA.

Animals↗
PubMed1988

Truncated variants of apolipoprotein B cause hypobetalipoproteinaemia.

Familial hypobetalipoproteinaemia is a rare autosomal dominant disorder in which levels of apo-B-containing plasma lipoproteins are approximately half-normal in heterozygotes and virtually absent in homozygotes. Here we describe mutations of the apo-B gene that cause two different truncated variants of apo-B in unrelated individuals with hypobetalipoproteinaemia. One variant, apo-B(His1795----Met-Trp-Leu-Val-Thr-Term) is predicted to be 1799 amino acids long and arises from deletion of a single nucleotide (G) from leucine codon 1794. This protein was found at low levels in very low density and low density lipoprotein fractions in the blood. The second, shorter variant, apo-B(Arg1306----Term), is caused by mutation of a CpG dinucleotide in arginine codon 1306 converting it to a stop codon and predicting a protein of 1305 residues. The product of this allele could not be detected in the circulation. The differences in size and behaviour of these two variants compared to apo-B100 or apo-B48 point to domains that may be important for the assembly, secretion or stability of apo-B-containing lipoproteins.

Amino Acid Sequence↗
PubMed1987

A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine.

Evidence suggests that intestinal apo-B48 is colinear with the amino-terminal half of hepatic apo-B100. To investigate the mechanism of apo-B48 production, we examined cDNA clones from human and rabbit small intestine. All clones contained a single C----T base difference from the hepatic sequence, resulting in a translational stop at codon 2153. Amplification by the polymerase chain reaction of cDNA from human and rabbit small intestine, rabbit liver, and the human hepatoma cell line HepG2 showed that the stop codon was only present in intestinal mRNA. Enterocyte genomic DNA did not contain the stop codon. We suggest that a co- or posttranscriptional C----U change may result in the production of apo-B48, which represents the amino-terminal 2152 amino acids of apo-B100. This is the first example of tissue-specific modification of a single mRNA nucleotide resulting in two different proteins from the same primary transcript.

Amino Acid Sequence↗