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D M Lilley

Publications and source records attributed to D M Lilley.

At least 91 records · Page 5Linked to original sources
PubMed1995

The global folding of four-way helical junctions in RNA, including that in U1 snRNA.

Helical junctions are important elements in the architecture of folded RNA molecules. The global geometry of fully base-paired four-way junctions between RNA helices has been analyzed by comparative gel electrophoresis. Junctions appear to fold by pairwise coaxial helical stacking in one of two possible stereochemically equivalent isomers based upon alternative selections of stacking partners. In the presence of 1 mM Mg2+, the two continuous helical axes are approximately at right angles to each other for all junctions studied, but the RNA junctions exhibit significant sequence-dependent differences in their structures as a function of ionic conditions. The four-way junction found in the U1 snRNA folded by coaxial helical stacking. It retained the 90 degrees crossed stacked structure under all ionic conditions tested, despite the presence of a G.A mismatch at the point of strand exchange.

Base Sequence↗
PubMed1995

The modular character of a DNA junction-resolving enzyme: a zinc-binding motif in bacteriophage T4 endonuclease VII.

Bacteriophage T4 endonuclease VII is one of a class of structure-selective enzymes that resolve helical branchpoints in DNA molecules. The sequence of this protein suggests a modular organisation. We have expressed a synthetic gene encoding endonuclease VII, which has been used in a directed mutagenesis exercise, with the aim of understanding the role of different sections of the protein sequence. Towards the N-terminal end of the protein lies a section of polypeptide in which four cysteine residues distributed in a CxxC--CxxC pattern co-ordinate one atom of zinc. The N-terminal section composed of amino acid residues 1 to 65 isolated from the remaining C-terminal section also binds one mole of zinc, suggesting that this region folds autonomously. Mutation shows that the outer cysteine residues are essential for zinc binding, while the inner cysteine residues are partially degenerate in that either one of the two (but not both) can be replaced while retaining some zinc. The activity as a junction-resolving enzyme correlated qualitatively with the presence of the zinc. In the C-terminal part of the protein lies a section that is 48% identical with a sequence found in the DNA repair protein T4 endonuclease V. We can replace the section of T4 endonuclease VII with the corresponding sequence from T4 endonuclease V with no change in the pattern of cleavage on four-way junctions. The evidence supports a modular construction for T4 endonuclease VII.

Amino Acid Sequence↗
PubMed1995

Two inequivalent folding isomers of the three-way DNA junction with unpaired bases: sequence-dependence of the folded conformation.

The global structures of three-way helical junctions in DNA that contain two unpaired bases at the point of strand exchange have been studied. All such junctions examined undergo a folding in the presence of metal ions, to a structure in which there is coaxial stacking of two helices, with the third at an acute angle. However there are potentially two isomers of this structure possible, which are stereochemically inequivalent. We find that the majority of sequences studied by us fold into the isomer in which the polarity of the bulge-containing strand passing from the unstacked to the stacked helix is 5' to 3'. However, some sequences adopt the alternative stacking isomer, in which the polarity of the bulged strand is 3' to 5'. These isomeric differences are in complete agreement with the findings from two other studies by NMR. In addition to the isomeric differences, we find significant sequence-dependent differences within series of junctions that adopt the same isomeric form in the presence of ions. We conclude that the folding of bulged three-way DNA junctions is highly sequence-dependent.

Base Sequence↗
PubMed1995

Binding of the junction-resolving enzyme bacteriophage T7 endonuclease I to DNA: separation of binding and catalysis by mutation.

Bacteriophage T7 endonuclease I is a resolving enzyme that selectively cleaves four-way DNA junctions, and related branched species. We have isolated mutants of this protein that retain full structural selectivity of binding to four-way junctions, but which are completely inactive as nucleases. This is consistent with a divisibility of structure-selective binding and catalysis. The mutations that inactivate endonuclease I as a nuclease are clustered into the second quarter of the primary sequence, a region that displays some sequence similarity with the related junction-resolving enzyme endonuclease VII from bacteriophage T4. This suggests that these residues may form the active site of these enzymes. The configuration of the helical arms of the junction bound by mutant endonuclease I has been investigated by gel electrophoretic methods. We find that the junction is bound in the presence or absence of magnesium ions, and that the global structure of the bound form is apparently identical with or without cations. The patterns of mobilities suggest that the structure of the junction becomes perturbed by the binding of the protein.

Amino Acid Sequence↗
PubMed1995

Structure of the four-way DNA junction and its interaction with proteins.

The four-way DNA junction is an important intermediate in recombination processes; it is, the substrate for different enzyme activities. In solution, the junction adopts a right-handed, antiparallel-stacked X-structure formed by the pairwise coaxial-stacking of helical arms. The stereochemistry is determined by the juxtaposition of grooves and backbones, which is optimal when the smaller included angle is 60 degrees. The antiparallel structure has two distinct sides with major and minor groove-characteristics, respectively. The folding process requires the binding of metal cations, in the absence of which, the junction remains extended without helix-helix stacking. The geometry of the junction can be perturbed by the presence of certain base-base mispairs or phosphodiester discontinuities located at the point of strand exchange. The four-way DNA junction is selectively cleaved by a number of resolving enzymes. In a number of cases, these appear to recognize the minor groove face of the junction and are functionally divisible into activities that recognize and bind the junction, and a catalytic activity. Some possible mechanisms for the recognition of branched DNA structure are discussed.

DNA↗
PubMed1995

Ionic interactions and the global conformations of the hammerhead ribozyme.

Here we investigate the global conformation of the hammerhead ribozyme. Electrophoretic studies demonstrate that the structure is folded in response to the concentration and type of ions present. Folding based on colinear alignment of arms II and III is suggested, with a variable angle subtended by the remaining helix I. In the probable active conformation, a small angle is subtended between helices I and II. Using uranyl photocleavage, an ion binding site has been detected in the long single-stranded region. The folded conformation could generate a preactivation of the scissile bond to permit in-line attack of the 2'-hydroxyl group, with a bound metal ion playing an integral role in the chemistry.

Base Sequence↗
PubMed1994

Modulation of tyrT promoter activity by template supercoiling in vivo.

We have found that initiation of RNA synthesis at the tyrT promoter of Escherichia coli can be stimulated on a plasmid by a factor of 4-6 by elevation of DNA supercoiling in vivo. Increased unconstrained plasmid supercoiling was achieved by inserting the tyrT promoter upstream of the tetracycline resistance gene tetA and transformation into a topA host. Under these conditions there is marked oversupercoiling of the plasmid DNA and we have shown previously that this can lead to increased promoter activity in the topological domain created. A critical element in the formation of this domain is the coupled transcription, translation and membrane insertion of tetA and we show that all of these events are important in the stimulation of tyrT promoter activity. The magnitude of the stimulation is in reasonable agreement with that measured in vitro as a function of plasmid supercoiling, if the unconstrained level of negative supercoiling in vivo is increased from a basal level of -sigma approximately 0.022 to -sigma approximately -0.052 by transcription-induced supercoiling. The induced supercoiling is very efficient, indicating that the tyrT promoter is itself contributing to the steady-state level despite a total lack of membrane anchorage for the tyrT transcription unit. This study provides a new example of the topological coupling of promoters.

Antiporters↗
PubMed1994

Homologous recombination. A ring for a warhead.

The active 'warhead' RuvB of the Escherichia coli protein that catalyzes the branch-migration step of homologous recombination is a helicase that binds DNA as a double-ring oligomer.

Bacterial Proteins↗
PubMed1994

Kinking of DNA and RNA helices by bulged nucleotides observed by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) has been used to demonstrate the bending of DNA and RNA helices for three series of double-stranded molecules containing bulge loops of unopposed adenosine nucleotides (An, n = 0-9). Fluorescein and rhodamine were covalently attached to the 5' termini of the two component strands. Three different methods were applied to measure the FRET efficiencies. The extent of energy transfer within each series increases as the number of bulged nucleotides varies from 1 to 7, indicating a shortening of the end-to-end distance. This is consistent with a bending of DNA and RNA helices that is greater for larger bulges. The FRET efficiency for DNA molecules with A9 bulges is lower than the efficiency for the corresponding A7 bulged molecules, although the A9 molecules exhibit increased electrophoretic retardation. Ranges of bending angles can be estimated from the FRET results.

Adenine Nucleotides↗
PubMed1994

Solution structure of a parallel-stranded tetraplex formed by d(TG4T) in the presence of sodium ions by nuclear magnetic resonance spectroscopy.

DNA sequences containing repetitive oligo-guanine sequences are observed in telomeric DNA and in recombination hotspots. In vitro, these sequences fold into at least two classes of unusual conformations involving the formation of guanine tetrads (G-quartets). Through the use of NMR, we have established that in solution in the presence of sodium ions, the oligodeoxynucleotide dTG4T forms a parallel-stranded tetraplex, with hydrogen-bonded guanine tetrads. Here we present the detailed evidence for this configuration in 100 mM sodium ions at neutral pH. We show that the parallel-stranded tetraplex structure is stabilised relative to the single-strands when Na ions are replaced by K ions, without any evidence of significant change in those chemical shifts associated with the tetraplex form. We also present a model for the Na+ solution structure of the tetraplex, by back-calculation from NOE volumes. We obtain excellent agreement with the data for structures with characteristics similar to B-DNA for the internal tetrads, G3 and G4. The outer tetrads, particularly the 5'-terminal G2, display some unstacking. We see no evidence for formation of thymine tetrads analogous to the uridine tetrads observed in RNA, but rather we find the terminal thymine conformations are not well determined by our data. We observe an uninterrupted cylindrical channel through the centre of the complex.

Cations, Monovalent↗
PubMed1994

Elevated unconstrained supercoiling of plasmid DNA generated by transcription and translation of the tetracycline resistance gene in eubacteria.

Our previous studies have indicated that the leu-500 promoter of Salmonella typhimurium is activated by local supercoiling arising from the transcription of a divergent promoter (Chen et al., 1992). For this to occur on a plasmid, we have shown that the transcribing RNA polymerase must be anchored to the cell membrane by transcription, translation, and export of the tetA gene and that the cell background must be topA. In this study we have used (AT)n reporter sequences to analyze changes in unconstrained supercoiling of plasmid DNA under the circumstances in which the leu-500 promoter becomes activated. (AT)n sequences undergo a structural transition to a cruciform at a threshold level of negative supercoiling that is determined by the length of the tract, and this can be detected in the cellular DNA by in situ chemical probing. These studies have shown that there is elevated unconstrained supercoiling in tetA-carrying plasmids in either Escherichia coli or S. typhimurium cells in exponential growth. This oversupercoiling depends on the function of the tetA gene in cis and the delta topA cell background. These are exactly the conditions that lead to the activation of the leu-500 promoter, supporting the proposed mechanism for the suppression of the leu-500 mutation by topA. Use of (AT)n sequences of different lengths has permitted us to estimate the extent of oversupercoiling. When the tetA gene was initiated using the strong tac promoter, we were able to detect increased unconstrained DNA supercoiling even in topA+ E. coli cells.

Base Sequence↗
PubMed1994

The high-resolution crystal structure of a parallel-stranded guanine tetraplex.

Repeat tracts of guanine bases found in DNA and RNA can form tetraplex structures in the presence of a variety of monovalent cations. Evidence suggests that guanine tetraplexes assume important functions within chromosomal telomeres, immunoglobulin switch regions, and the human immunodeficiency virus genome. The structure of a parallel-stranded tetraplex formed by the hexanucleotide d(TG4T) and stabilized by sodium cations was determined by x-ray crystallography to 1.2 angstroms resolution. Sharply resolved sodium cations were found between and within planes of hydrogen-bonded guanine quartets, and an ordered groove hydration was observed. Distinct intra- and intermolecular stacking arrangements were adopted by the guanine quartets. Thymine bases were exclusively involved in making extensive lattice contacts.

Computer Graphics↗
PubMed1994

Large-scale opening of A + T rich regions within supercoiled DNA molecules is suppressed by salt.

Large-scale cooperative helix opening has been previously observed in A + T rich sequences contained in supercoiled DNA molecules at elevated temperatures. Since it is well known that helix melting of linear DNA is suppressed by addition of salt, we have investigated the effects of added salts on opening transitions in negatively supercoiled DNA circles. We have found that localised large-scale stable melting in supercoiled DNA is strongly suppressed by modest elevation of salt concentration, in the range 10 to 30 mM sodium. This has been shown in a number of independent ways: 1. The temperature required to promote cruciform extrusion by the pathway that proceeds via the coordinate large-scale opening of an A + T rich region surrounding the inverted repeat (the C-type pathway, first observed in the extrusion of the ColE1 inverted repeat) is elevated by addition of salt. The temperature required for extrusion was increased by about 4 deg for an addition of 10 mM NaCl. 2. A + T rich regions in supercoiled DNA exhibit hyperreactivity towards osmium tetroxide as the temperature is raised; this reactivity is strongly suppressed by the addition of salt. At low salt concentrations of added NaCl (10 mM) we observe that there is an approximate equivalence between reducing the salt concentration, and the elevation of temperature. Above 30 mM NaCl the reactivity of the ColE1 sequences is completely supressed at normal temperatures. 3. Stable helix opening transitions in A + T rich sequences may be observed with elevated temperature, using two-dimensional gel electrophoresis; these transitions become progressively harder to demonstrate with the addition of salt. With the addition of low concentrations of salt, the onset of opening transitions shifts to higher superhelix density, and by 30 mM NaCl or more, no transitions are visible up to a temperature of 50 degrees C. Statistical mechanical simulation of the data indicate that the cooperativity free energy for the transition is unaltered by addition of salt, but that the free energy cost for opening each basepair is increased. These results demonstrate that addition of even relatively low concentrations of salt strongly suppress the large-scale helix opening of A + T rich regions, even at high levels of negative supercoiling. While the opening at low salt concentrations may reveal a propensity for such transitions, spontaneous opening is very unlikely under physiological conditions of salt, temperature and superhelicity, and we conclude that proteins will therefore be required to facilitate opening transitions in cellular DNA.

Base Composition↗
PubMed1994

Structure of four-way DNA junctions containing a nick in one strand.

We have investigated the structure of the four-way helical DNA junction containing a single covalent discontinuity (nick) in one strand. These could result from either unitary strand exchange processes, or the action of nucleases upon a complete junction. We have employed gel electrophoresis methods to study the global configuration of arms in these junctions. We find that the junction carrying a nick in one strand undergoes a folding process in the presence of magnesium ion concentrations greater than 200 microM. Comparison of the electrophoretic mobilities of the six possible derivative junctions with two long and two shortened arms suggests that the folding occurs by coaxial stacking of pairs of helical arms, which is supported by the suppression of reactivity to osmium tetroxide of thymine bases at the centre of the junction. However, unlike the complete junction (i.e. the junction without nicked strands), the two stacked pairs of helices lie at a mutual angle of approximately 90 degrees. The folding process generates two kinds of strands; two continuous strands and two exchanging strands. Two isomers of the right-angled stacked structure are possible, depending on the selection of stacking partners; it appears that the critical factor determining the relative stabilities of these isomers is the location of the nick. Thus the nicked junctions fold into the isomer that locates the nick on the exchanging strand. However, if the nick is not located at the point of strand exchange, the junction reverts to the stacked X-structure of the complete junction, even if the nick is moved by a single base-pair. These results suggest that the exchanging strands may be significantly strained in the structure of the complete four-way junction, such that an interruption to the continuity at this position allows the two stacked helices to disengage, and rotate to an angle where the overall electrostatic repulsion may be lower.

Base Sequence↗