Search PubMed⌕ Search

Biomedical subjects

M J Waring

Publications and source records attributed to M J Waring.

At least 109 records · Page 6Linked to original sources
PubMed1987

Interaction of nucleosome core particles with distamycin and echinomycin: analysis of the effect of DNA sequences.

Two fragments of Xenopus borealis DNA 135 and 189 base-pairs long were separately incorporated into nucleosome core particles by reconstitution with chicken erythrocyte histones, and incubated with echinomycin (a bis-intercalating antitumor antibiotic) or distamycin (a minor groove-binding, non-intercalating antibiotic). Controlled digestion of these defined sequence core particles using DNAase I revealed new cleavage products, indicative of a change in orientation of the DNA molecule on the surface of the nucleosome. This new rotational setting of DNA within the core particle induced by antibiotic binding appears to be practically independent of DNA sequence, although some differences were noted between the patterns of fragments observed in the various experiments, most likely reflecting the exact number and disposition of the antibiotic binding sites.

Animals↗
PubMed1987

Footprinting at low temperatures: evidence that ethidium and other simple intercalators can discriminate between different nucleotide sequences.

Footprinting experiments employing DNAase I have been performed at 4 degrees C. At this temperature several simple intercalating ligands, including both ethidium and proflavine, can be seen to induce marked changes in the pattern of cleavage. From an analysis of the changes in patterns of digestion by DNAase I we deduce that ethidium binds best to regions of mixed nucleotide sequence, especially those containing alternating purines and pyrimidines. Binding seems to be weakest to poly dA sequences which consequently appear as regions of relatively enhanced cleavage. Attempts to reproduce these changes using DNAase II as a footprinting tool were unsuccessful.

Base Composition↗
PubMed1987

Analysis of the effects of antibiotics on the structure of nucleosome core particles determined by DNAase I cleavage.

Patterns of sensitivity to DNAase I cleavage have been analysed in order to investigate the effects of anti-tumour antibiotics and related drugs on nucleosome core particles containing different DNA restriction fragments. In this article, we review the experimental results which show that after controlled digestion of defined-sequence core particles, new cleavage products appear in the enzyme digestion patterns which lie approximately mid-way between the strong bands characteristic of native nucleosome core particles. The effects of the antibiotics, which include bis-intercalators as well as minor groove-binding ligands (but not monofunctional intercalators), are explained in terms of an induced change in rotational setting (phasing) of the core DNA. The new rotational positioning of DNA induced by antibiotic binding appears to be almost independent of DNA sequence, although some differences can be observed with the various pieces of DNA, most likely reflecting the exact number and disposition of antibiotic binding sites.

Anti-Bacterial Agents↗
PubMed1986

Antibiotics which can alter the rotational orientation of nucleosome core DNA.

Four well-characterised DNA-binding ligands have been tested for effects on reconstituted nucleosome core particles containing the 160 bp tyrT DNA fragment. Two, netropsin and berenil, were found to change the rotational orientation of the DNA on the surface of the protein as judged by marked alterations in the pattern of fragments produced by exposure to DNAase I. Qualitatively their effects were very similar to those previously reported for the related antibiotic distamycin, suggesting that the phenomenon of induced rotation may be a characteristic property of ligands which bind in the narrow groove of the DNA helix. Two intercalators did not produce the effect but, at high concentrations, caused gross disruption of the nucleoprotein structure with apparent release of DNA from the histone octamer. At moderate concentrations little or no effect was detectable with nogalamycin, suggestive of failure to bind as a result of constraints on local opening of the DNA helix. With moderate concentrations of actinomycin, protection of GpC sequences was clearly visible together with some evidence of increase in helix pitch, but no sign of altered phasing of DNA within the nucleosome core particles.

Anti-Bacterial Agents↗
PubMed1986

Echinomycin and distamycin induce rotation of nucleosome core DNA.

When nucleosome cores reconstituted from chicken erythrocyte histones and a 160 bp DNA molecule are exposed to echinomycin, a bis-intercalating antitumour antibiotic, the DNA appears to rotate with respect to the histone octamer by about half a turn. New bands appear in patterns of DNAase I digestion at positions approximately mid-way between those characteristic of control core samples, while the control pattern is largely suppressed. Similar (but not identical) changes are produced when nucleosome cores are exposed to distamycin, a non-intercalating DNA-binding antibiotic. The effects of both ligands can be explained in terms of a change in rotational orientation of the core DNA, so as to place antibiotic binding sites on the inward-facing (concave) surface of the DNA supercoil. Presumably this serves to optimise non-bonded contacts with the polynucleotide backbone. These results establish that the positioning of DNA about the histone octamer is not absolutely determined by its nucleotide sequence, but may be modified by the binding of such relatively small molecules as antibiotics.

Animals↗
PubMed1986

Nucleotide sequence binding preferences of nogalamycin investigated by DNase I footprinting.

Four DNA restriction fragments, designated tyrT, pTyr2, pUC13, and Xbs1, have been used as substrates for footprinting studies with DNase I in the presence of the anthracycline antibiotic nogalamycin. With each fragment a distinct pattern of antibiotic-protected binding sites is observed, but no concensus sequence emerges from the data. All sites are located in regions of alternating purine-pyrimidine sequence, most commonly associated with the dinucleotide steps TpG (CpA) and GpT (ApC), suggesting that the preferred binding sites may contain all four nucleotides and/or that peculiarities of the dynamics of DNA conformation at alternating sequences may be critical for nogalamycin binding. Some concentration dependence of footprinting patterns is evident, in contrast to previous studies with a variety of sequence-specific ligands. Enhanced susceptibility to attack by DNase I is commonly observed at sequences flanking strong antibiotic-binding sites. Nogalamycin selectively inhibits cleavage of DNA at certain guanine-containing sequences by the G-specific photosensitized reaction with methylene blue. Comparison of these effects with its action on the G-specific reaction with dimethyl sulfate suggests that the amino sugar moiety of nogalamycin may be preferentially located in the minor helical groove at some binding sites but in the major groove at others.

Anthracyclines↗
PubMed1986

DNA sequence preferences for the anti-cancer drug mitoxanthrone and related anthraquinones revealed by DNase I footprinting.

The interaction has been studied of several anthraquinone-based intercalating drugs, including the anti-cancer agent mitoxantrone, with defined sites of DNA. A 160 base pair DNA sequence from tyrT was employed for footprinting with DNase I. The anthraquinones had aminoalkylamino substituents in various positions of the ring system. Inhibition of enzymatic cutting of the DNA was observed at various positions on the sequence, mostly around some of the pyrimidine-3',5'-purine sites. Enhancements to cutting were observed clustered around AT-rich regions. The compounds showed differences in detailed footprinting behaviour, which have been related to differences in their mode of interaction with DNA as found in earlier computer modelling studies.

Anthraquinones↗
PubMed1986

Footprinting reveals that nogalamycin and actinomycin shuffle between DNA binding sites.

The hypothesis that sequence-selective DNA-binding antibiotics locate their preferred binding sites by a process involving migration from nonspecific sites has been tested by footprinting with DNAase I. Footprinting patterns on the tyrT DNA fragment produced by nogalamycin and actinomycin change with time after mixing the antibiotic with the DNA. Sites of protection as well as enhanced cleavage are seen to develop in a fashion which is both temperature and concentration-dependent. At certain sites cutting is transiently enhanced, then blocked. Limited evidence for slow reaction with echinomycin and mithramycin is presented, but the kinetics of footprinting with daunomycin and distamycin appear instantaneous. The feasibility of adducing direct evidence for shuffling by footprinting seems to be governed by slow dissociation of the antibiotic-DNA complex. It may also be dependent upon the mode of binding, be it intercalative or non-intercalative in character.

Binding Sites↗
PubMed1986

DNA sequence recognition by under-methylated analogues of triostin A.

Two new analogues of TANDEM (des-N-tetramethyl triostin A) have been synthesised in an effort to elucidate the molecular basis of DNA nucleotide sequence recognition in this series of compounds. Their binding preferences have been investigated by DNAase I footprinting and differential inhibition of restriction nuclease attack. The presence of a single N-methyl group on only one valine residue (in [N-MeVal4] TANDEM) abolishes the ability to recognise DNA, presumably because this antibiotic analogue has suffered an unfavourable conformational change in the depsipeptide ring. A bis-methylated analogue, [N-MeCys3, N-MeCys7]TANDEM, was found to interact quite strongly with DNA and afforded binding sites, rich in AT residues, identical to those of TANDEM. Footprinting with various DNA fragments of known sequence showed that this analogue recognises sequences containing the dinucleotide TpA, although we cannot exclude the possibility that it binds to ApT as well. [N-MeCys3, N-MeCys7]TANDEM inhibits cutting by RsaI, a restriction enzyme that recognises GTAC but not by Sau3AI which recognises GATC. This provides further supportive evidence that the ligand (and, by extension, TANDEM itself) prefers binding to sequences containing the dinucleotide step TpA.

Amino Acid Sequence↗
PubMed1986

Studies on antibiotic biosynthesis by protoplasts and resting cells of Streptomyces echinatus. Part II. Effect of chromophore precursors.

Washed cell and protoplast suspensions from Streptomyces echinatus A8331, which produces the quinoxaline antibiotic echinomycin, have been used to study the effects of analogues of the natural chromophore upon antibiotic biosynthesis. Addition of quinoline-2-carboxylic acid caused a decrease in the labelling of echinomycin from L-[methyl-14C]methionine and an increase in labelled chloroform-extractable material. Quinoxaline-2-carboxylic acid increased the incorporation of radioactivity into both fractions. Thieno[3,2-b]pyridine-5-carboxylic acid, 6-methylquinoline-2-carboxylic acid, and quinoline-2-carboxylic acid (also to a lesser extent 7-chloroquinoxaline-2-carboxylic acid) increased markedly the incorporation of radioactivity into chloroform-extractable material and virtually abolished echinomycin synthesis. Autoradiographs of extracts from suspensions supplemented with the latter four analogues revealed bis-substituted metabolites not found in unsupplemented cultures. When protoplast suspensions were incubated with L-[U-14C]serine, L-[U-14C]valine, or DL-[benzene ring-U-14C]tryptophan, quinoline-2-carboxylic acid, thieno[3,2-b]pyridine-5-carboxylic acid, and 6-methylquinoline-2-carboxylic acid directed the synthesis of antibiotically active bis derivatives at the expense of echinomycin. When analogues of quinoxaline-2-carboxylic acid previously found unsuitable for incorporation by growing cultures were tested in protoplast suspensions, only isoquinoline-3-carboxylic acid caused a large increase in the incorporation of radioactivity from L-[methyl-14C]methionine into chloroform-extractable material. With DL-[benzene ring-U-14C]tryptophan as the radiolabel, benzotriazoline-2-acetic acid and 6-bromoquinoxaline-2-carboxylic acid as well as isoquinoline-3-carboxylic acid sharply reduced the labelling of echinomycin.

Amino Acids↗
PubMed1986

Overview of the interaction between chemotherapeutic agents and DNA.

Some chemotherapeutic agents, such as the antibiotics mitomycin and bleomycin, modify the structure of DNA by chemical reactions involving the formation or breakage of covalent bonds. Others interact with the macromolecule reversibly to form a transient complex which may be intercalative or nonintercalative in character. Techniques are available to probe the extent of perturbation of DNA structure produced by these drugs, and they reveal subtle differences between the effects of various ligands. In recent years bifunctional intercalating agents such as the quinoxaline antibiotics have been discovered; their binding to DNA is often tighter than seen with simple (monofunctional) intercalators and there is evidence for nucleotide sequence-selectivity. Footprinting experiments have been employed to identify preferred ligand-binding sites in natural DNA fragments (CpG sequences in the case of echinomycin) and have revealed that local perturbations of the helical structure can be propagated into DNA regions flanking the antibiotic-binding sites. Crystallographic evidence suggests that echinomycin and its congeners recognise GC base-pairs by hydrogen bonding between the carbonyl groups of alanine residues in the antibiotic and the 2-amino groups of guanine nucleotides in the minor groove of the DNA helix. Kinetic studies support the hypothesis that sequence-selective antibiotic molecules "shuffle" between different binding sites in the process of locating their optimal (preferred) sites.

Animals↗
PubMed1986

DNA sequence selectivity of three biosynthetic analogues of the quinoxaline antibiotics.

Mono- and bis-quinoline analogues of echinomycin, and a bis-3-amino-quinoxaline analogue of triostin A, have been prepared by directed biosynthesis and investigated for sequence selectivity in binding to DNA. Binding isotherms for bis-3-amino triostin A interacting with four natural and two synthetic DNA species have been determined by using solvent-partition analysis with radiolabelled antibiotic. They reveal a similar pattern of preferences to that seen with the parent compound: tighter binding occurs with the more guanine and cytosine (GC)-rich DNAs and in every case the association constant is increased two- to five-fold over the value recorded for triostin A. Deoxyribonuclease I footprinting patterns measured for the quinoline analogues of echinomycin differ from those observed with the parent antibiotic in that an additional strong site of protection occurs around the CpG sequence at position 35 in the tyrT fragment. Footprints for bis-3-amino triostin A reveal a substantially more selective pattern of cleavage inhibition than seen with the natural antibiotics: only two or three distinct binding sites are identified in tyrT DNA and four in pTyr2 DNA. Each is centred around one or more CpG steps, but many more CpG-containing sequences are unprotected. The analogue seems to prefer CpG steps flanked by at least one adenine and thymine (AT) pair, optimally ACGN. Enhancement of cutting at AT-rich sequences surrounding their binding sites is seen with all three of the new antibiotics. The results lend weight to the idea that novel sequence selectivity can be attained by making appropriate substitution on the chromophores of quinoxaline antibiotics.

Anti-Bacterial Agents↗
PubMed1985

Kinetics of dissociation of nogalamycin from DNA: comparison with other anthracycline antibiotics.

Stopped-flow spectrometry and simple mixing techniques have been employed to investigate the detergent-induced dissociation of anthracycline antibiotics from natural and synthetic DNAs. Both daunomycin and nogalamycin dissociate more slowly from poly(dG-dC) than from poly(dA-dT) but the difference is much more marked for nogalamycin. With an equimolar mixture of poly(dG-dC) and poly(dA-dT), or with poly(dA-dC).poly(dG-dT), dissociation of nogalamycin occurs very slowly. In all cases the release of antibiotic from a synthetic polynucleotide is a one-step process following a single exponential. Dissociation of daunomycin, adriamycin and iremycin from calf thymus DNA is a more complex reaction which requires a two-exponential fit, in contrast to earlier reports, but differences between the behaviour of the three antibiotics are minor. Dissociation of nogalamycin from natural DNA requires a three-exponential fit, is in general far slower, and depends upon the base composition, the level of binding and the time allowed for the complex to equilibrate. It is concluded that sequence selectivity is minimal or lacking for daunomycin, whereas nogalamycin binding is sequence dependent and probably involves migration of the antibiotic between DNA binding sites. There is an inverse correlation between dissociation rate constants and antibacterial potency in simple tests.

Animals↗
PubMed1985

Kinetic evidence that echinomycin migrates between potential DNA binding sites.

The hypothesis that echinomycin locates its preferred nucleotide sequences in DNA by a process of "shuffling" between potential binding sites has been tested. Immediately after reacting with calf thymus DNA the antibiotic is relatively weakly bound inasmuch as the complex dissociates quite rapidly when detergent is added. If the complex is allowed to equilibrate for various periods of time after mixing, an increasing proportion of the bound antibiotic dissociates slowly on addition of detergent. The kinetics of appearance of the slowly-dissociating form, and its dependence upon ionic strength, are fully consistent with the shuffling model. In contrast the dissociation profiles from poly(dG-dC) and poly(dA-dT) are independent of mixing time.

Base Sequence↗
PubMed1985

Incorporation of fluorotryptophan into triostin antibiotics by Streptomyces triostinicus.

The quinoxaline chromophores of the antibiotics produced by Streptomyces triostinicus are derived from tryptophan. Protoplasts of this organism made novel products when they were incubated with DL-5-fluorotryptophan or DL-6-fluorotryptophan. When added to batch cultures of the organism, DL-5-fluorotryptophan, at concentrations as low as 10 microM, inhibited both mycelial growth and triostin production, but gave rise to novel products. These have been characterized, using fast atom bombardment mass spectrometry, as novel triostins in which one or both of the quinoxaline rings contain an atom of fluorine. The chromatographic properties of the triostins arising from the incorporation of DL-5-fluorotryptophan are very similar to those of triostins containing chlorine or bromine at position 6 of the quinoxaline ring; they are clearly different from those having a chlorine atom at position 7. Accordingly, it is suggested that the carbon atom at position 5 of the indole ring of tryptophan ends up at position 6 of the quinoxaline ring system in triostins A and C.

Anti-Bacterial Agents↗
PubMed1984

DNA structural variations produced by actinomycin and distamycin as revealed by DNAase I footprinting.

The technique of DNAase I footprinting has been used to investigate preferred binding sites for actinomycin D and distamycin on a 160-base-pair DNA fragment from E. coli containing the tyr T promoter sequence. Only sites containing the dinucleotide step GpC are protected by binding of actinomycin, and all such sites are protected. Distamycin recognizes four major regions rich in A + T residues. Both antibiotics induce enhanced rates of cleavage at certain regions flanking their binding sites. These effects are not restricted to any particular base sequence since they are produced in runs of A and T by actinomycin and in GC-rich sequences by distamycin. The observed increases in susceptibility to nuclease attack are attributed to DNA structural variations induced in the vicinity of the ligand binding site, most probably involving changes in the width of the helical minor groove.

Base Sequence↗
PubMed1984

Kinetic evidence for redistribution of actinomycin molecules between potential DNA-binding sites.

The kinetics of interaction between actinomycin D and DNA have been measured by stopped-flow and detergent-dissociation methods. The results are consistent with a model in which the antibiotic initially binds to many sequences on the heterogeneous DNA lattice and subsequently 'shuffles' between the available sites until a thermodynamically determined optimal state of binding is attained. The amplitudes of the two slowest components in the reaction with calf thymus DNA do not vary in parallel as the total level of antibiotic binding is increased; they appear to reflect directly the redistribution of antibiotic molecules along the DNA lattice. The dissociation profile is shown to depend upon the time for which the antibiotic and DNA are premixed, so that for short mixing times a higher proportion of the decay is represented by faster-dissociating species. The rate of appearance of the slowest-dissociating species correlates well with the slowest optical change in the association reaction. Stopped-flow experiments indicate that the antibiotic first binds to sites on natural DNA with an average association constant of 4 X 10(3) M-1 and that it subsequently migrates to sites with higher affinity. Similar experiments performed with poly(dG-dC) are less easily interpreted and seem to indicate that conformational changes or cooperative effects can also occur.

Binding Sites↗