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D M Lilley

Publications and source records attributed to D M Lilley.

At least 163 records · Page 9Linked to original sources
PubMed1988

Helix stability and the mechanism of cruciform extrusion in supercoiled DNA molecules.

The kinetic properties of cruciform extrusion in supercoiled DNA molecules fall into two main classes. C-type cruciforms extrude in the absence of added salt, at relatively low temperatures, with large activation energies, while S-type cruciforms exhibit no extrusion in the absence of salt, and maximal rates at 50 mM NaCl, with activation energies about one quarter those of the C-type. These diverse properties are believed to reflect two distinct pathways for the extrusion process, and are determined by the nature of the sequences which form the context of the inverted repeat. C-type kinetics are conferred by A + T rich sequences, implying a role of helix stability in the selection. In this study we have shown that: 1. Helix-destabilising solvents (dimethyl formamide and formamide) facilitate extrusion by normally S-type molecules at low temperatures in the absence of salt. 2. C-type extrusion is strongly suppressed by low concentrations (2-4 microM) distamycin, at which concentrations S-type extrusion is enhanced. 3. Some extrusion occurs in a C-type construct in the presence of 50 mM NaCl. This is increased by addition of 3 microM distamycin, under which conditions extrusion becomes effectively S-type. Thus S-type constructs can behave in a quasi-C-type manner in the presence of helix-destabilising solvents, and C-type extrusion is suppressed by binding a compound which stabilises A + T rich regions of DNA. Helix destabilisation leads to C-type behaviour, while helix stabilisation results in S-type properties. These studies demonstrate the influence of contextual helix stability on the selection of kinetic mechanism of cruciform extrusion.

Bacteriocin Plasmids↗
PubMed1988

DNA supercoiling and the leu-500 promoter mutation of Salmonella typhimurium.

DNA supercoiling is an important, but relatively poorly understood factor which influences promoter function. leu-500 is a point mutation in the promoter of the leucine operon of Salmonella typhimurium which confers leucine auxotrophy. It can be phenotypically suppressed by mutations in the topA gene, which encodes topoisomerase I, implicating DNA supercoiling in the regulation of this promoter. We have demonstrated that phenotypic suppression of this mutant promoter is transcriptional, and that topA mutations restore function to the mutant promoter. Transcription from the leu-500 promoter was examined in a series of strains harbouring topA and tos (presumptive gyr) mutations, each of which exhibits a different level of in vivo plasmid supercoiling. Promoter function did not correlate with the level of supercoiling but rather with the presence or absence of a functional topA gene. Furthermore, when cloned onto a multicopy plasmid, the leu-500 promoter failed to function, even in a topA background. Thus, local rather than global changes in DNA topology are implicated in the activation of this promoter.

Bacterial Proteins↗
PubMed1987

The mechanism of cruciform formation in supercoiled DNA: initial opening of central basepairs in salt-dependent extrusion.

There are two alternative pathways by which inverted repeat sequences in supercoiled DNA molecules may extrude cruciform structures, called C-type and S-type. S-type cruciforms, which form the great majority, are characterised by absolute requirement for cations to promote extrusion, which then proceeds at higher temperatures and with lower activation parameters than for C-type cruciforms. The mechanism proposed for S-type extrusion involves an initial opening of basepairs limited to the centre of the inverted repeat, formation of intra-strand basepairing and a four-way junction, and finally branch migration to the fully extruded cruciform. The model predicts that central sequence changes will be more kinetically significant than those removed from the centre. We have studied the kinetics of cruciform extrusion by a series of inverted repeats related to that of pIRbke8 by either one or two mutations in the symmetric unit. We find that mutations in the central 8 to 10 nucleotides may profoundly affect extrusion rates--the fastest being 2000-fold faster than the slowest, whereas mutations further from the centre affect rates to a much smaller extent, typically up to ten-fold. These data support the proposed mechanism for extrusion via central opening.

Base Sequence↗
PubMed1987

A two-state conformational equilibrium for alternating (A-T)n sequences in negatively supercoiled DNA.

We have made a study of the pattern of osmium tetroxide modification in supercoiled plasmids containing alternating (A-T)n tracts. Two distinct alternative patterns may be obtained, depending upon conditions. At moderate salt concentrations, or at low temperature, only thymine bases close to the centres of the tracts were modified, consistent with the presence of a cruciform structure. At higher temperatures in the absence of cations, uniform modification throughout the tracts was observed. The cationic concentration required to stabilize cruciform structure depends markedly on its charge, and a number of transition metal ions were totally ineffective. The results are interpreted in terms of a two-state equilibrium between the cruciform and a perturbed helical structure, the position of which is temperature- and salt-dependent. For longer (A-T)n tracts, a third pattern of osmium tetroxide modification is found at intermediate salt concentrations, consistent with a cruciform having an extensively disrupted four-way junction.

Adenine↗
PubMed1987

The anomalous gel migration of a stable cruciform: temperature and salt dependence, and some comparisons with curved DNA.

We have made an analysis of the gel electrophoretic properties of a pseudo-cruciform fragment, a linear DNA molecule containing a stable cruciform. The migration of this construct was analysed in polyacrylamide gels at a various temperatures in the range 5 degrees to 55 degrees C, and in the presence of NaCl, MgCl2 or ethidium bromide. The magnitude of the anomalous migration (retardation) was almost temperature independent up to 40 degrees C, but decreased strongly beyond this point, extrapolating to normal migration at 70 degrees C. Addition of salts reduced the anomaly. This took the form of a continuous reduction in anomalous migration with the addition of NaCl up to 60 mM, while with MgCl2 there was a sharp reduction in the anomaly to a constant value which is reached by 10 mM. Under these conditions, moreover, the migration of the fragment became almost temperature-independent over the entire range. These results have been interpreted to reflect the influence of ion binding at the four-way junction on the relative disposition of the cruciform arms. The detailed electrophoretic properties of the pseudo-cruciform are in marked contrast to those of sequence-directed curved DNA fragments. In particular, the response to the addition of 1 microgram/ml ethidium bromide offers a convenient method for distinguishing between anomalous retardation arising from curvature (greatly reduced anomaly) or a cruciform junction (enhanced anomaly).

Coliphages↗
PubMed1987

Influence of cation size and charge on the extrusion of a salt-dependent cruciform.

We have made a comparative study of the kinetics of cruciform extrusion by a salt-dependent (S-type) cruciform in the presence of a variety of metal ions and related species. We find that the nature of the cation present has a marked effect on the observed kinetics, and that different cations differ greatly in the efficiency with which they promote the extrusion process. We can divide the ions into four classes according to the optimal ionic concentration required for maximal extrusion rate. Group Ia cations and tetramethyl ammonium are most effective in promoting extrusion at 50 to 60 mM. Group IIa and selected transition metal ions (notably manganese) are effective over a wide range, extending down to 200 microM. Hexaminecobalt(III) and the polyamines promote extrusion at concentrations as low as 15 to 40 microM. Most remaining ions examined, including trivalent ions such as Al(III) and many transition metal ions are totally ineffective. Within the first two groups, we observe a marked correlation between the rate of cruciform extrusion promoted and ionic radius, the larger ions giving faster extrusion rates. We interpret this to indicate specific ion binding occurring in the transition state of the extrusion process. We may rationalize all the data in terms of a model for salt-dependent cruciform extrusion, in which the transition state resembles a partially extruded protocruciform. This creates an anionic "cavity" with selective ion binding properties, and its stabilization is therefore ion size-dependent.

Anions↗
PubMed1986

(A-T)n tracts embedded in random sequence DNA--formation of a structure which is chemically reactive and torsionally deformable.

Alternating d(A-T)n sequences which are contiguous with DNA of effectively random sequence have an abnormal conformation in linear DNA molecules. These regions are strongly reactive towards chemical modification by osmium tetroxide, and are preferentially cleaved by micrococcal nuclease. Both the chemical modification and the enzymic cutting occur uniformly through the alternating tract, and there is no evidence for enzyme or chemical sensitivity in the interfaces between the tract and DNA of normal conformation. These reactivities have a requirement for an alternating sequence. In addition to chemical reactivity, alternating (A-T)n sequences exhibit anomalously small twist changes on cruciform formation, suggesting that the pre-extruded DNA is underwound. We propose that the alternating sequences adopt an altered conformation which is subject to easy torsional deformation.

Base Sequence↗
PubMed1986

A dominant influence of flanking sequences on a local structural transition in DNA.

We have discovered a striking dependence of a structural transition in DNA on sequences that are distanced from those directly participating in the transformation. The dominant factor determining the selection of kinetic properties of cruciform extrusion is the sequence of the DNA that flanks the inverted repeat. The sequence of the inverted repeat itself appears to have little or no influence. The critical sequences that confer the unusual kinetics exhibited by the ColE1 cruciform are very A+T-rich. A single such sequence is sufficient, which may be as short as 100 bp, and it can control inverted repeats placed at either end. The effects operate in cis, are independent of polarity, and may be effective over relatively long distances. The influence of context has wide implications, possibly including the control of gene expression.

Bacteriocin Plasmids↗
PubMed1986

Highly selective chemical modification of cruciform loops by diethyl pyrocarbonate.

Diethyl pyrocarbonate reacts with the single-stranded loops of cruciform structures with great selectivity. Adenine bases are carbethoxylated, as a result of which the backbone may be cleaved with piperidine, and the level of chemical modification at each base may be determined. We have studied the ColE1 and (A-T)34 cruciforms of pColIR315 and pXG540. In each case we observe maximal modification at the most central adenosine of the loop, and an overall pattern of modification corresponding to a total loop size of about six bases. The results may be interpreted in terms of a model in which the loop has a defined tertiary structure. No modification was detected at either cruciform four-way junction, suggesting that this region is fully base-paired.

Base Sequence↗
PubMed1986

The structure of cruciforms in supercoiled DNA: probing the single-stranded character of nucleotide bases with bisulphite.

The single-stranded character of cytosine bases in three cruciform structures has been assessed by an examination of reactivity towards sodium bisulphite. Unpaired cytosine residues undergo deamination at C4 to give deoxyuracil, and propagation in an ung Escherichia coli host results in C-G----T-A transition mutations, detectable by restriction cleavage or sequence analysis. Very high frequencies of such mutations have been found at cruciform loops, confirming their unpaired character, with almost zero background mutation frequencies elsewhere. A low level of modification was observed at the four-way junction of a cruciform. The results indicate that the optimal cruciform loop size is four bases, with loose 'breathing' at the first base pair at the top of the cruciform stem at 37 degrees C, and little or no opening of base pairs at the four-way junction.

Base Sequence↗
PubMed1986

Stress-induced cruciform formation in a cloned d(CATG)10 sequence.

The synthetic alternating purine-pyrimidine sequence, d(CATG)10.d(CATG)10, has been cloned into a 2.079-kb pBR322-derived plasmid (pLN1) and its conformation studied under torsional stress. The resultant plasmid, pLNc40, is hypersensitive to cleavage by the single strand-specific nucleases, S1 nuclease and Bal31 nuclease, and to modification by the single strand-selective reagent, osmium tetroxide. The S1-hypersensitive site of this plasmid predominates over those previously mapped in pBR322. Site-specific cleavage of pLNc40 with the resolvase T4 endonuclease VII demonstrates that this alternating purine-pyrimidine tract selectively forms a cruciform structure when stably integrated into a negatively supercoiled plasmid. Quantitative measurements of the twist change (-4.3 +/- 0.2) and free energy of formation (16.2 +/- 0.5 kcal/mol) of this cruciform have been made from two-dimensional gel electrophoresis experiments, and correspond well with the predicted values of cruciform formation for this sequence. We conclude that cruciform extrusion versus the B-Z transition is the favoured conformation of this insert under torsional stress.

Base Sequence↗
PubMed1986

DNA supercoiling.

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Anti-Bacterial Agents↗
PubMed1986

Cyclic adduct formation at structural perturbations in supercoiled DNA molecules.

Supercoiled DNA can stabilize the existence of micropolymorphic structures, which may be sequence dependent. A good example is cruciform formation from inverted repeat sequences. DNA sequences exhibiting a high degree of two-fold symmetry, for instance, the 13-bp inverted repeat of ColE1, can adopt cruciform geometry when supercoiled, as judged by sensitivity to single-strand-specific nucleases and to resolvase and by topological changes revealed by gel electrophoresis. The present report shows that these structures are also excellent substrates for the local formation of cyclic adducts by bromoacetaldehyde, glyoxal or osmium tetroxide. Individual reagents may show pronounced differences in sequence selectivity, although all appear to react strongly at the ColE1 sequence. The reactions initially occur at the centre of the inverted repeats; this is followed by a conformational change such that the modification propagates outwards from the centre. These compounds constitute a valuable addition to the list of probes available for the investigation of local perturbations in DNA structure.

Acetaldehyde↗
PubMed1985

Facile cruciform formation by an (A-T)34 sequence from a Xenopus globin gene.

We have studied the structure adopted by an (A-T)34 sequence from a Xenopus globin gene when present in a negatively supercoiled plasmid. A variety of enzyme and chemical probing experiments and electrophoretic migration shift methods reveal that the sequence adopts cruciform geometry at moderate levels of supercoiling. The structure has the lowest free energy of formation yet observed for a cruciform, and no detectable kinetic barrier preventing rapid interconversion between extruded and unextruded conformations. Analysis of band-shift experiments reveals a twist change on cruciform formation of -5.8, slightly smaller than the -6.5 we would predict on the basis of a transition from B DNA. An attractive explanation consistent with this discrepancy is that the (A-T)34 stretch is locally underwound to about 11.7 base-pairs/helical turn at low levels of supercoiling. This calculation is made on the assumption that the cruciform junction is structurally similar to those examined previously, which is supported by the nuclease digestion results. This perturbed helical structure could be of considerable biological significance.

Acetaldehyde↗
PubMed1985

The kinetic properties of cruciform extrusion are determined by DNA base-sequence.

The extrusion kinetics of two cruciforms derived from unrelated DNA sequences differ markedly. Kinetic barriers exist for both reactions, necessitating elevated temperatures before extrusion proceeds at measureable speeds, but the dependence upon temperature and ionic strength is quite different for the two sequences. One, the ColE1 inverted repeat, exhibits a remarkably great temperature dependence of reaction rate and is suppressed by moderate amounts of NaCl or MgCl2. In contrast, the other, a synthetic inverted repeat present in pIRbke8, shows more modest temperature dependence and has a requirement for the presence of salt, with optimal concentrations being 50 mM NaCl or 100 microM MgCl2. Under optimal conditions, cruciform extrusion rates are fast (t1/2 less than 60m) at 37 degrees C for both sequences at native superhelix densities. In 50 mM NaCl the pIRbke8 inverted repeat is characterised by an Arrhenius activation energy of 42.4 +/- 3.2 kcal mole -1. The differences in kinetic properties between the two sequences indicate that DNA base sequence is itself an important factor in determining cruciform kinetics, and possibly even in the selection of the mechanistic pathway.

Base Sequence↗
PubMed1984

Thermodynamics of the ColE1 cruciform. Comparisons between probing and topological experiments using single topoisomers.

The sensitivity of the ColE1 cruciform to four enzyme and chemical probes of secondary structure has been studied as a function of plasmid topology. Purified topoisomers of pColIR515 have been probed with S1 nuclease, Bal31 nuclease, phage T4 endonuclease VII or osmium tetroxide, and site-specific reaction quantified. Closely similar profiles of reactivity as a function of linking difference were obtained for each probe. Electrophoresis of the pure topoisomers on polyacrylamide/agarose gels revealed a discontinuity in migration as a function of linking difference. Above a threshold linking difference, topoisomers exhibit pronounced reduction in mobility. The linking difference at which this band shift is found correlates precisely with that required for site-specific reaction with the four probes. We conclude that both probing and topological methods are valuable in the study of cruciform structure in supercoiled DNA. The band shift has been measured with accuracy to allow the calculation of the twist change that accompanies the transition, corresponding to delta Tw = -3.2 +/- 0.1. Using this value together with the critical linking difference we calculate a free energy of formation for this structure delta G = 18.4 +/- 0.5 kcal mol-1 (1 kcal = 4.184 kJ).

DNA Topoisomerases, Type I↗