The locus of sequence-directed and protein-induced DNA bending

The bending locus of trypanosome kinetoplast DNA, identified by gel electrophoresis, has tracts of a simple repeat sequence (CA5-6 T) symmetrically distributed about it, with a repeat interval of 10 base pairs. The analogous bending induced when catabolite gene activating protein binds to its recognition sequence near the promoter of the Escherichia coli lac operon is centred on a site about 5-7 base pairs away from the centre of the protein binding site.     

DNA bend direction by phase sensitive detection

Gel electrophoresis of DNA and protein-DNA complexes has been a key method used in studies of sequence-directed and protein-induced DNA bending. Natural DNA sequences can have protein binding sites adjacent to A-tract bending sites, resulting in the potential for the formation of topologically complex shapes in a localized DNA regulatory domain. An essential first step in deducing the structure and functional significance of such domains is elucidation of the relative direction of bending, which can be determined from the electrophoretic mobilities of isomers having varied helical phasing between the bends. Taking DNA bent around CAP protein as a standard, we conclude that the junction bending model correctly predicts the direction of bending at A tracts in kinetoplast DNA. The overall direction of the bend is towards the minor groove at the centre of the A tract.     

Importance of DNA stiffness in protein-DNA binding specificity

From the first high-resolution structure of a repressor bound specifically to its DNA recognition sequence it has been shown that the phage 434 repressor protein binds as a dimer to the helix. Tight, local interactions are made at the ends of the binding site, causing the central four base pairs (bp) to become bent and overtwisted. The centre of the operator is not in contact with protein but repressor binding affinity can be reduced at least 50-fold in response to a sequence change there. This observation might be explained should the structure of the intervening DNA segment vary with its sequence, or if DNA at the centre of the operator resists the torsional and bending deformation necessary for complex formation in a sequence dependent fashion. We have considered the second hypothesis by demonstrating that DNA stiffness is sequence dependent. A method is formulated for calculating the stiffness of any particular DNA sequence, and we show that this predicted relationship between sequence and stiffness can explain the repressor binding data in a quantitative manner. We propose that the elastic properties of DNA may be of general importance to an understanding of protein-DNA binding specificity.     

Ribozyme recognition of RNA by tertiary interactions with specific ribose 2'-OH groups.

Shortened forms of the group I intron from Tetrahymena catalyse sequence-specific cleavage of exogenous oligonucleotide substrates. The association between RNA enzyme (ribozyme) and substrate is mediated by pairing between an internal guide sequence on the ribozyme and a complementary sequence on the substrate. RNA substrates and cleavage products associate with a binding energy greater than that of base-pairing by approximately 4 kcal-mol-1 (at 42 degrees C), whereas DNA associates with an energy around that expected for base-pairing. It has been proposed that the difference in binding affinity is due to specific 2'-OH groups on an RNA substrate forming stabilizing tertiary interactions with the core of the ribozyme, or that the RNA.RNA helix formed upon association of an RNA substrate and the ribozyme might be more stable than an RNA.DNA helix of the same sequence. To differentiate between these two models, chimaeric oligonucleotides containing deoxynucleotide residues at successive positions along the chain were synthesized, and their equilibrium binding constants for association with the ribozyme were measured directly by a new gel electrophoresis technique. We report here that most of the extra binding energy can be accounted for by discrete RNA-ribozyme interactions, the 2'-OH group on the sugar residue three nucleotides from the cleavage site contributing the most interaction energy. Thus, in addition to the well documented binding of RNA to RNA by base-pairing, 2'-OH groups within a duplex can also mediate association between RNA molecules.     

DNA cleavage catalysed by the ribozyme from Tetrahymena.

An RNA enzyme derived from the self-splicing intervening sequence of Tetrahymena thermophila catalyses sequence-specific cleavage of an oligodeoxyribonucleotide substrate. Compared with RNA, the DNA substrate is bound very weakly and is cleaved very slowly, revealing the importance of the RNA 2'-hydroxyl group in both the binding and chemical steps. The finding that catalysis by RNA can extend to DNA substrates indicates new possibilities for the transposition of intervening sequences and for the design of DNA cleavage agents with novel sequence specificities.     

An unusual DNA structure detected in a telomeric sequence under superhelical stress and at low pH

Telomeric sequences of DNA, which are found at the ends of linear chromosomes, have been attracting attention as potential sites for the formation of unusual DNA structures. They consist of (GnTm) or (GnATm) motifs (n greater than or equal to m) and, in the single-stranded state, form hairpins stabilized by non-canonical G.G pairs. In the duplex state and under superhelical stress they exhibit hypersensitivity to SI nuclease which by analogy with homopurine-homopyrimidine sequences may reflect the formation of an unusual structure. To determine whether this is the case we have inserted into a plasmid the Tetrahymena telomeric motif (G4T2).(A2C4) and probed it by two-dimensional gel electrophoresis, chemical modification and oligonucleotide binding. Our data demonstrate that, under superhelical stress and at low pH, the insert does indeed adopt a novel DNA conformation. We have concluded that in this structure the C-rich strand forms a hairpin stabilized by non-Watson-Crick base pairs C.C+ and A.A+, whereas the G-rich strand remains unstructured. We term this new DNA structure the (C,A)-hairpin.     

Chemical probing of homopurine-homopyrimidine mirror repeats in supercoiled DNA

We have recently shown that under superhelical stress and/or acid pH the homopurine-homopyrimidine tracts conforming to the mirror symmetry (H palindromes) form a novel DNA structure, the H form. According to our model, the H form includes (1) a triplex formed by half of the purine strand and by the homopyrimidine hairpin and (2) the unstructured other half of the purine strand. We used four specially designed sequences to demonstrate that, in accordance with our model, the mirror symmetry is essential for facile formation of the H form detected by two-dimensional gel electrophoresis. Here we report that, under conditions favouring the H-form extrusion, guanines of the 3' half of the purine strand are protected against alkylation by dimethylsulphate, whereas adenines of the 5' half of the purine strand react with diethyl pyrocarbonate. These data indicate that the 3' half of the homopurine strand is within the triplex whereas the 5' half is unstructured, in full agreement with our model.     

Measurement of anomalously high hydration of (dA)n.(dT)n double helices in dilute solution

Different DNA sequences have different physical properties, which seem to be important for their biological function. In particular, (dA)n.(dT)n has many unusual features, which include resistance to conformational changes in a variable chemical environment, an unusual thermodynamics of interaction with ligands, and the inability to reassociate into nucleosomes. Short A.T base-pair runs also play a critical role in DNA bending. It is believed that hydration of DNA is an important factor in determining the physical chemical and biological properties of different regions of DNA. Until now, however, it has not been possible to study the details of the hydration of DNA in dilute solution with sufficient sensitivity and precision. Moreover, it was not known if different base sequences differ in the extent of their hydration. Indirect evidence that (dA)n.(dT)n can be hydrated to a greater extent than other DNA sequences may be inferred from a recent study of the binding of drugs to polynucleotides. Here we used a novel high-precision technique measuring ultrasonic velocity to obtain direct estimates of the extent of hydration of various oligo- and polynucleotides in dilute solution. We report that different DNA sequences differ in their hydration, and that (dA)n.(dT)n in particular has an anomalously high level of hydration.     

Hypervariable ultra-long telomeres in mice

Telomere structure and behaviour is less well understood in vertebrates than it is in ciliates and yeasts (reviewed in ref. 1). Like all other eukaryotic chromosomes, those of vertebrates terminate in an array of a short repeated sequence. In vertebrates this sequence is (TTAGGG)n, as shown by in situ hybridization. In humans, these terminal repeats are heterogeneous in length, averaging about 10 kilobases in blood cells. Here we report the structure and inheritance of the terminal repeats present at mouse telomeres. The (TTAGGG)n tracts are many times larger than those present at human telomeres. Because of their constancy in length through somatic cell divisions, they are resolved as multiple discrete restriction fragments of up to 150 kilobases. Strikingly, this banding pattern is highly polymorphic within populations of inbred mice, suggesting an unusually high mutation rate. Indeed, although the banding pattern is inherited in a largely mendelian fashion, (TTAGGG)n tracts of new size appear frequently in family studies.     

Is there left-handed DNA at the ends of yeast chromosomes?

Tracts of the alternating copolymer poly(dGdT . dCdA) have been observed in a variety of eukaryotes. Such tracts are of particular interest since homopolymers of this sequence can exist in vitro as left-handed Z form DNA. We have found that the yeast Saccharomyces cerevisiae contains at least 30 poly(GT) tracts at dispersed genomic locations. We show here that one subset of these tracts is located at the ends (telomeres) of the yeast chromosome. In addition, we show that poly(dGdT . dCdA) tracts are added to the ends of the extrachromosomal ribosomal DNA molecules of Tetrahymena when cloned in yeast. These data represent the first reported association between a homopolymeric sequence and a chromosome structure.     

Uncoupling of initiation site cleavage from subsequent headful cleavages in bacteriophage T1 DNA packaging

The packaging of intracellular DNA into heads is a key feature in the morphogenesis of bacteriophage particles. In many phages a performed empty head precursor, the prohead, is filled with DNA from a concatemeric substrate consisting of tandemly repeated genome lengths. The addition of outer shell proteins completes head formation. The DNA molecules released from particles of the coliphage T1 exist as three major permutations of nucleotide sequence. Such limited permutation can be explained by the modification of Streisinger's 'headful' mechanism proposed for phage P22. DNA packaging is initiated at a specific site (the pac site) on the concatemeric precursor. While this site is cleaved, subsequent cleavages (headful cleavages) are dependent only on head-filling and are not defined in terms of nucleotide sequence. Headfuls of DNA, consisting of slightly more than a genome length, are packaged in three successive cycles of head-filling to produce the permuted and terminally redundant molecules characteristic of T1 DNA. To elucidate the regulation of this process, we have studied the DNA metabolism of T1 head mutants. We describe here the properties of a mutant in gene 13.3 which is defective for headful cleavage but remains proficient in pac site cleavage. The observation in this mutant that concatemers are degraded to unit-length molecules by repeated pac site cleavage suggests a model of headful packaging in which pac site initiation and processive head-filling compete for the DNA substrate.     

Sequence-specific artificial photo-induced endonucleases based on triple helix-forming oligonucleotides

Homopyrimidine oligonucleotides bind to homopurine-homopyrimidine sequences of duplex DNA forming a local triple helix. This binding can be demonstrated either directly by a footprinting technique, gel assays, or indirectly by inducing irreversible reactions in the target sequence, such as photocrosslinking or cleavage. Binding occurs in the major groove with the homopyrimidine oligonucleotide orientated parallel to the homopurine strand. Thymine and protonated cytosine in the oligonucleotide form Hoogsteen-type hydrogen bonds with A.T and G.C Watson-Crick base pairs, respectively. Here we report that an 11-residue homopyrimidine oligonucleotide covalently attached to an ellipticine derivative by its 3' phosphate photo-induces cleavage of the two strands of a target homopurine--homopyrimidine sequence. To our knowledge, this is the first reported case of a sequence-specific artificial photoendonuclease. In addition we show that a strong binding site for a free ellipticine derivative is induced at the junction between the triplex and duplex structures on the 5' side of the bound oligonucleotide. On irradiation, cleavage is observed on both strands of DNA. This opens new possibilities for inducing irreversible reactions on DNA at specific sites by the synergistic action of a triple helix-forming oligonucleotide and an intercalating agent.     

Protection against UV-induced pyrimidine dimerization in DNA by triplex formation

Cyclobutane and [6-4]-pyrimidine dimers are major photoproducts of ultraviolet-irradiated DNA. The yield of these photoproducts is dependent on the sequence and structure of the DNA. By analysing the photofootprints of fragments produced by cleavage of the DNA chain near [6-4]-pyrimidine dimers, we show here that a homopurine-homopyrimidine insert (with either d(TC)x or d(C)n) in plasmid pUC19 is, as expected, a good target for UV-induced pyrimidine-dimer formation. But we find that dimerization is virtually completely suppressed when the pyrimidine oligonucleotides d(TC)y or d(C)m are added to DNA carrying d(TC)x- or d(C)n-containing inserts, respectively. This effect is dependent on the type of oligonucleotide used and is site-specific. The protection occurs under acidic conditions that favour the formation of intermolecular triplexes between the homopurine-homopyrimidine inserts and homologous oligopyrimidines. We therefore conclude that triplex formation effectively protects the DNA duplex from UV-induced damage (pyrimidine dimerization). This observation makes the photofootprinting assay a very promising method for studying intermolecular and intramolecular triplexes (H-form DNA) both in vitro and in vivo.     

Poly(dA).poly(dT) is a B-type double helix with a distinctively narrow minor groove

The structure of poly(dA).poly(dT) currently arouses great interest, mainly because dAn.dTn stretches are associated with considerable DNA bending. Until recently the heteronomous DNA described by Arnott et al., with the poly(dA) and poly(dT) chains in A and B conformations respectively, was the only detailed model of this structure. Following our earlier studies of the interaction of DNA and monovalent ions, we examined the X-ray diffraction of the bivalent Ca2+ salt of poly(dA).poly(dT) (Ca-poly(dA).poly(dT)) and found no sign of a heteronomous structure: Ca-poly(dA).poly(dT) in fibres shows fully equivalent B-type conformations of the opposite sugar-phosphate chains. A revision of the structure of the sodium salt, Na-poly(dA).poly(dT), based on this result, yields only a slightly heteronomous structure with each chain in a B-type conformation, which is in much better agreement with the experimental data underlying the original heteronomous model. Both structures, Ca- and Na-poly(dA).poly(dT), have a minor groove narrower than that of the B form: this peculiarity seems to be very important for the interaction of poly(dA).poly(dT) and biologically significant molecules (including proteins and antibiotics). The specific base-pair positions in poly(dA).poly(dT) may account for the DNA bending adjacent to dAn.dTn tracts.     

Programmed cell death in Laminaria japonica (Phaeophyta) tissues infected with alginic acid decomposing bacterium

TdT-mediated dUTP-biotin nick end labeling (TUNEL) is a sensitive and valid method for detecting DNA cleavage in programmed cell death (PCD). Using this method, DNA cleavage was observed in Laminaria japonica sporophytic tissues, which were infected with alginic acid decomposing bacterium. It was found that DNA cleavage occurred 5 min after the infection, the fragments with 3′-OH groups of cleaved nuclear DNA increased with time of infection and spread from the infection site. Although no typical DNA ladder (200 bp/180 bp) was detected by routine agarose gel electrophoresis, the cleavage of nuclear DNA fragments of 97～48.5 kb could be detected by pulsed field gel electrophoresis (PFGE). By using CaspGLOWTM fluorescein active caspase-3 staining method, caspase-3 activity has been detected in response to the infection of alginic acid decomposing bacterium. Our results are similar to the observations in hypersensitive response (HR) of higher plant, suggesting that the rapid cell death of L. Japonica infected by alginic acid decomposing bacterium might be involved in PCD, and indicating that the occurrence of PCD is an active defense process against the pathogen's infection.     

Model for antenatal diagnosis of beta-thalassaemia and other monogenic disorders by molecular analysis of linked DNA polymorphisms

Polymorphisms of DNA restriction sites within the human fetal globin genes have been used to identify chromosomes that carry beta-thalassaemia genes in individuals heterozygous for this disease. This has allowed an antenatal diagnosis for beta-thalassaemia to be carried out by observation of the pattern of the inherited polymorphism of a linked DNA sequence not involved in the genetic pathogenesis of the disease. In the populations we have investigated there is no constant pattern of polymorphism that segregates with the beta-thalassaemia gene. The use of linked polymorphisms should, therefore, be applicable to antenatal diagnosis both of beta-thalassaemia and of any other single-gene defect for which there is a DNA probe specific for a sequence linked to the affected locus.     

The structure of an oligo(dA).oligo(dT) tract and its biological implications

Poly(dA).poly(dT) has unusual properties in that it cannot associate into nucleosomes and short, phased runs of it cause DNA bending. The crystal structure of a B-type DNA dodecamer containing a homopolymeric run of six A.T base pairs shows that this region possesses special structural features, including a system of bifurcated hydrogen bonds, which explains some of the properties of this simple homopolymer.