Purine-pyrimidine Sequences Research Articles

Circular dichroism of poly(dG-dC) modified by the carcinogens N-methyl-4-aminoazobenzene or 4-aminobiphenyl.

Poly(dG-dC) was modified to different extents by the carcinogens 4-aminobiphenyl (ABP) or N-methyl-4-aminoazobenzene (MAB). HPLC analysis of the enzymatically hydrolyzed modified polymers indicates that more than 90% of the ABP and 81% of the MAB modification occurs at the C8 position of guanine. The conformational changes of the unmodified and modified polymers were studied as a function of ethanol and magnesium ion concentrations by the use of circular dichroism (CD). The modified polymers show a CD inversion pattern similar to that of the salt-induced B to Z transition of poly(dG-dC). Both of the modified polymers require less salt or ethanol than the unmodified polymer for the inversion of the spectra. The amount of ethanol or magnesium needed to induce the inverted CD spectrum is inversely proportional to the percentage of bound ABP or MAB. These data indicate that ABP and MAB can enhance conversion from B to Z conformation in alternating purine-pyrimidine sequences.

DNA conformation is determined by economics in the hydration of phosphate groups.

Mixed sequence DNA can exist in two right-handed and one left-handed double helical conformations--A, B and Z. Under conditions of high water activity the B conformation prevails. If the water activity is reduced on addition of salt or organic solvents, transformation occurs to A-DNA or, in DNAs with alternating purine-pyrimidine sequences, to the left-handed Z-DNA. In crystal structure analyses of oligonucleotides, the free oxygen atoms of adjacent phosphate groups along the polynucleotide chain in B-DNA are found at least 6.6 A apart and individually hydrated whereas they are as close as 5.3 A in A-DNA and 4.4 A in Z-DNA, and bridged by water molecules. We suggest that this more economical hydration in A- and Z-DNA compared with B-DNA is the underlying cause of B----A and B----Z transitions.

Efficient transcription of a Caenorhabditis elegans heat shock gene pair in mouse fibroblasts is dependent on multiple promoter elements which can function bidirectionally.

A divergently transcribed pair of Caenorhabditis elegans hsp16 genes was introduced into mouse fibroblasts by stable transfection with vectors containing bovine papillomavirus plasmid maintenance sequences and a selectable gene. The hsp16 genes were transcriptionally inactive in the mouse cells under normal growth conditions and were strongly induced by heat shock or arsenite. In a cell line with 12 copies of the gene pair, there were estimated to be more than 10,000 hsp16 transcripts in each cell after 2 h of heat shock treatment. The hsp16 transcript levels were more than 100 times higher than those of a gene with a herpes simplex virus thymidine kinase gene promoter carried on the same vector. A single heat shock promoter element (HSE) could activate bidirectional transcription of the two hsp16 genes when placed between the two TATA elements, but the transcriptional efficiency was reduced 10-fold relative to that of the wild-type gene pair. Four overlapping HSEs positioned between the two TATA elements resulted in inducible bidirectional transcription at greater than wild-type levels. The number of HSEs can therefore be a major determinant of the promoter strength of heat-inducible genes in mammalian cells. Partial disruption of an alternating purine-pyrimidine sequence between the two hsp16 genes had no significant effect on their transcriptional activity.

Efficient transcription of a Caenorhabditis elegans heat shock gene pair in mouse fibroblasts is dependent on multiple promoter elements which can function bidirectionally.

A divergently transcribed pair of Caenorhabditis elegans hsp16 genes was introduced into mouse fibroblasts by stable transfection with vectors containing bovine papillomavirus plasmid maintenance sequences and a selectable gene. The hsp16 genes were transcriptionally inactive in the mouse cells under normal growth conditions and were strongly induced by heat shock or arsenite. In a cell line with 12 copies of the gene pair, there were estimated to be more than 10,000 hsp16 transcripts in each cell after 2 h of heat shock treatment. The hsp16 transcript levels were more than 100 times higher than those of a gene with a herpes simplex virus thymidine kinase gene promoter carried on the same vector. A single heat shock promoter element (HSE) could activate bidirectional transcription of the two hsp16 genes when placed between the two TATA elements, but the transcriptional efficiency was reduced 10-fold relative to that of the wild-type gene pair. Four overlapping HSEs positioned between the two TATA elements resulted in inducible bidirectional transcription at greater than wild-type levels. The number of HSEs can therefore be a major determinant of the promoter strength of heat-inducible genes in mammalian cells. Partial disruption of an alternating purine-pyrimidine sequence between the two hsp16 genes had no significant effect on their transcriptional activity.

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.

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.

A left-handed (Z) conformation of poly(dA-dC).poly(dG-dT) induced by polyamines.

Blocks of potential Z-DNA forming alternating purine-pyrimidine (APP) sequences are widely dispersed in native DNAs. We have studied the effects of naturally occurring polyamines on the conformation of a synthetic APP sequence, poly(dA-dC).poly(dG-dT) by circular dichroism spectroscopy. In the presence of micromolar concentrations of spermidine (125 microM) and spermine (16 microM), this polymer undergoes B to Z transition in low ionic strength (2 mM Na+) buffers. The concentration of polyamines required for B to Z transition increases with Na+ in the buffer and a straight line is obtained on plotting ln[Na+] vs. ln [spermidine 3+]. However, at concentrations of polyamines higher than those necessary to induce B to Z transition, Z-DNA converts to psi-DNA, an ordered, twisted, tight packing arrangement of the double helix. These results suggest a pathway for the transient formation of Z-DNA segments in vivo by interaction of the ubiquitous polyamines with naturally occurring blocks of APP sequences.

Sequence-dependent energetics of the B-Z transition in supercoiled DNA containing nonalternating purine-pyrimidine sequences.

The likelihood that a given DNA sequence will adopt the Z conformation in negatively supercoiled DNA depends on the energy difference between the B form and the Z form for that sequence relative to other sequences in the same molecule. This energy can be viewed simply as a sum of energies for the nearest-neighbor interactions within the sequence plus the energy required to stabilize the B-Z boundaries. Knowledge of these energetic terms would be of value in predicting when sequences become left-handed in response to negative superhelicity. Here we present an approach that can be used to determine the free-energy changes associated with all the nearest-neighbor interactions that can occur in Z-DNA. Synthetic stretches of d(C-G)n containing one or two transversions were cloned into plasmids, and the extent of the B-Z transition as a function of negative superhelicity was determined for each insert by two-dimensional agarose gel electrophoresis. By subjecting the data to statistical mechanical analysis, it was possible to evaluate the energetic penalty resulting from each base-pair (bp) substitution. Guanine to cytosine transversions cost 2.4 kcal (1 cal = 4.18 J)/(mol X bp), whereas guanine to thymine transversions cost 3.4 kcal/(mol X bp), to stabilize in the Z conformation. We have used these numbers, along with energetic values determined by others for the B-Z transition, to predict that certain strictly nonalternating purine and pyrimidine sequences may adopt the Z form readily.

Long-range allosteric effects on the B to Z equilibrium by daunomycin

Spectroscopic and fluorometric methods were used to study the binding of the anticancer drug daunomycin to poly[d(G-C)] and poly[d(G-m5C)] under a variety of solution conditions. Under high-salt conditions that favor the left-handed Z conformation, binding isotherms for the interaction of the drug with poly[d(G-C)] are sigmoidal, indicative of a cooperative binding process. Both the onset and extent of the cooperative binding are strongly dependent upon the ionic strength. The binding data may be explained by a model in which the drug preferentially binds to B-form DNA and acts as an allosteric effector on the B to Z equilibrium. At 2.4 M NaCl, binding of as little as one drug molecule per 20 base pairs (bp) results in the conversion of poly[d(G-C)] from the Z form entirely to the B form, as inferred from binding data and demonstrated directly by circular dichroism measurements. Similar results are obtained for poly[d(G-m5C)] in 50 mM NaCl and 1.25 mM MgCl2. Under these solution conditions, it is possible to demonstrate the Z to B structural transition in poly[d(G-m5C)] as a function of bound drug by the additional methods of sedimentation velocity and susceptibility to DNase I digestion. The transmission of allosteric effects over 20 bp is well beyond the range of the drug's binding site of 3 bp. Since daunomycin preferentially binds to alternating purine-pyrimidine sequences, which are the only sequences capable of the B to Z transition, the allosteric effects described here may be of importance toward understanding the mechanism by which the drug inhibits DNA replicative events.(ABSTRACT TRUNCATED AT 250 WORDS)

Z-DNA forms without an alternating purine-pyrimidine sequence in solution.

Nuclear magnetic resonance spectra (proton and phosphorus-31) and ultraviolet absorption spectra of the DNA decamer d(br5CGbr5CGATbr5CGbr5CG), in which the central two adenine-thymine base pairs are out of order with the rest of the purine-pyrimidine alternation sequence, indicate that under appropriate solvent conditions (high salt and methanol) the molecule undergoes a structural transition from a right-handed B-DNA conformation to a left-handed Z-DNA conformation. Measurements of the two-dimensional nuclear Overhauser effect on the decamer indicate that all of the guanines as well as the two equivalent thymines adopt the syn conformation.

Conformational variability of poly(dA-dT).poly(dA-dT) and some other deoxyribonucleic acids includes a novel type of double helix.

The article reviews data indicating that poly(dA-dT).poly(dA-dT) is able of adopting three distinct double helical structures in solution, of which only the A form conforms to classical notions. The other two structures have dinucleotides as double helical repeats. At low salt concentrations poly(dA-dT).poly(dA-dT) adopts a B-type alternating conformation which is exceptionally variable. Its architecture can gradually move in the limits demarcated by the CD spectra with inverted long wavelength CD bands and the 31P NMR spectra with a very low and a 0.6 ppm separation of two resonances. Contrary to Z-DNA, the 31P NMR spectrum of the limiting alternating B conformation of poly(dA-dT).poly(dA-dT) is characterized by an upfield shift of one resonance. We attribute the exceptional conformational flexibility of the alternating B conformation to the unequal tendency of bases in the dA-dT and dT-dA steps to stack. However, by assuming the limiting alternating B conformation, the variability of the synthetic DNA is not exhausted. Specific agents make it isomerize into another conformation by a fast, two-state mechanism, which is reflected by a further deepening of the negative long wavelength CD band and a downfield shift of the 31P NMR resonance of poly(dA-dT).poly(dA-dT) that was constant in the course of the gradual alterations of the alternating B conformation. These changes are, however, qualitatively different from the way poly(dG-dC).poly(dG-dC) behaves in the course of the B-Z isomerization. Poly(dG-dC).poly(dG-dC) displays purine-pyrimidine (dGpdC) resonance in the characteristic downfield position, while the downfield resonance of poly(dA-dT).poly(dA-dT) belongs to the pyrimidine-purine (dTpdA) phosphodiester linkages. Consequently, phosphodiester linkages in the purine-pyrimidine steps play a similar role in the appearance of the Z form to the pyrimidine-purine phosphodiesters in the course of the isomerization of poly(dA-dT).poly(dA-dT). This excludes that the high-salt structures of poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) are members of the same conformational family. We call the high-salt conformation of poly(dA-dT).poly(dA-dT) X-DNA. It furthermore follows from the review that synthetic molecules of DNA with alternating purine-pyrimidine sequences of bases can adopt either the Z form or the X form, or even both, depending on the environmental conditions. This introduces a new dimension into the DNA double helix conformational variability. The possible biological relevance of the X form is suggested by experiments with linear molecules of natural DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of rat ribosomal DNA II: Identification of the highly repetitive DNA in the 3′ non-transcribed spacer

The nucleotide sequence of one of the non-transcribed spacer subclones, p1.7, from the region 3′ to rat 45 S pre-rRNA has been determined. Within 1612 base-pairs, the fragment contains two distinct regions of highly repetitive DNA, one of which can serve as a site for initiation in vitro by RNA polymerase III. The first is the alternating purine-pyrimidine sequence (A-C) 21. The second of these regions has 95% homology to the identifier sequence and served as the template for RNA polymerase III transcription in vitro. The in vitro polymerase III template is aligned in opposite polarity to the direction of transcription of 45 S rRNA. Located near the identifier sequence is a region that is 59% homologous to the type-II Alu sequences. It would seem, therefore, that members of more than one highly repetitive sequence family have accumulated in the non-transcribed spacers. These data also suggest that within the non-transcribed spacers these families have evolved (sequence variation) at different rates, until one of them, the Alu type-II-like element, may represent a new Alu type-II subfamily.

Left handed DNA in synthetic and topologically constrained form V DNA and its implications in protein recognition

We have investigated structural transitions in Poly(dG-dC) and Poly(dG-Me5dC) in order to understand the exact role of cations in stabilizing left-handed helical structures in specific sequences andthe biological role, if any, of these structures. From a novel temperature dependent Z ⇌ B transition it has been shown that a minor fluctuation in Na+ concentration at ambient temperature can bring about B to Z transition. Forthe first time, wehave observed a novel Z⇌B⇌Zuble transition in poly(dG-Me5dC) as the Na+ concentration is gradually increased. This suggests that a minor fluctuation in Na+ concentration in conjunction with methylation may transform small stretches of CG sequences from one conformational state to another. These stretches could probably serve as sites for regulation. Supercoiled formV DNA reconstituted from pBR322 and pβG plasmids have been studied as model systems, in order to understand the nature and role of left-handed helical conformation in natural sequences. A large portion of DNA in form V, obtained by reannealing the two complementary singlestranded circles is forced to adopt left-handed double helical structure due to topological constraints (Lk = 0). Binding studies with Z-DNA specific antibody and spectroscopic studies confirm the presence of left-handed Z-structure in the pβG and pβR322 form V DNA. Cobalt hexamine chloride, which induces Z-form in Poly(dG-dC) stabilizes the Z-conformation in form V DNA even in the non-alternating purine-pyrimidine sequences. A reverse effect is observed with ethidium bromide. Interestingly, both topoisomerase I and II (from wheat germ) act effectively on form V DNA to give rise to a species having an electrophoretic mobility on agarose gel similar to that of open circular (form II) DNA. Whether this molecule is formed as a result of the left-handed helical segments of form V DNA undergoing a transition to the right-handed B-form during the topoisomerase action remains to be solved.

Z-DNA in the rat somatostatin gene.

Two alternating purine-pyrimidine tracts flank the rat gene encoding the polypeptide hormone somatostatin. One lies 5' and one 3' to the gene; both consist of tandem repeats of the dinucleotide TG, with 25 repeats in the tract 5' and 15 in the tract 3' to the gene, and both are bordered on at least one edge by short repeated sequences. Characterization of supercoiled plasmids containing these sequences reveals that both form Z-DNA. Using S1 nuclease as a probe of DNA conformation we have investigated the fine structure of the Z-DNA and have shown: 1) that the entire Z-DNA segment as well as the single-stranded junctions flanking it is sensitive to S1 nuclease; 2) that the B-DNA/Z-DNA junction can be contained within the ends of the alternating purine-pyrimidine tract; and 3) that the sequences bordering the alternating purine-pyrimidine tracts affect the extent of Z-DNA propagation, sometimes as a result of their own apparently nonB-DNA conformation. We have also examined the published sequence of the human somatostatin gene (Shen, L.-P., and Rutter, W.J. (1984) Science 224, 168-171) for alternating purine-pyrimidine or potential Z-DNA-forming sequences and compared them to those present in the rat gene. We find that the human gene contains a 32-base pair alternating purine-pyrimidine sequence in an analogous position to the (TG)25 tract 5' to the rat gene, although the two sequences are not homologous. There are also six shorter alternating purine-pyrimidine elements 5' to the transcribed sequences which are positioned almost identically in the two genes with respect to the transcription initiation sites, although their sequences are not well conserved. We propose that the parallel placement of alternating purine-pyrimidine or potential Z-DNA-forming sequences 5' to the somatostatin genes from two species is a result of structural, in contrast to sequence, conservation. These observations suggest that the rat somatostatin gene may be a good model system for the investigation of the function of Z-DNA in the regulation of gene expression.

Crystal structure of Z-DNA without an alternating purine-pyrimidine sequence.

In left-handed Z-DNA, consecutive nucleotides along the chain alternate in the syn and anti conformations. Purine residues form the syn conformation readily and up to now all Z-DNA crystal structures have sequences of alternating purines and pyrimidines. However, we find that d(C-G-A-T-C-G) with the cytosines brominated or methylated on C-5 crystallizes as Z-DNA. The structure reveals thymines in syn and adenines in anti conformations. This suggests that Z-DNA may occur in sequences other than those with alternating purine-pyrimidine sequence.

Selective binding of the estradiol receptor to a region at least one kilobase upstream from the rat prolactin gene.

The interaction of the estradiol receptor with cloned DNA fragments from the prolactin gene was investigated using a competitive binding assay. A DNA fragment from the 5'-flanking region of the rat prolactin gene was able to bind the estradiol receptor selectively. DNA fragments representing most of the remaining 10 kb of the prolactin gene showed little or no selective binding to the estradiol receptor. The fragment from the 5'-flanking region which selectively binds the receptor is located between 1.2 and 2.0 kb upstream from the transcription initiation site rather than in a more proximal position as has been observed for the interaction of several other steroid hormone receptors with specific genes. Nucleotide sequence analysis demonstrated that this region of DNA contains two large segments of alternating purine-pyrimidine sequence. One of the alternating purine-pyrimidine regions is very large, containing more than 160 nucleotides of almost perfect poly(dG-dT). Further studies will be required to determine if the receptor interacts specifically with these interesting sequences. However, the ability of the estradiol receptor to bind to an upstream 5'-flanking region of the prolactin gene may be part of the mechanism which allows the receptor to stimulate the transcription of this gene selectively.

Conformations of alternating purine-pyrimidine DNAs in high-CsF solutions and their reversal by dipyrandium, ethidium and high temperature.

Chiroptical properties of synthetic DNAs with alternating purine-pyrimidine sequences were studied in high-CsF solutions. It was found that this salt transformed all the DNAs to non-canonical conformations like Z or X, which show striking negative bands in the long wavelength part of the CD spectra. The negative bands were reversed upon interaction with dipyrandium, ethidium and at high temperature.

Sequence-dependent changes in the chiroptical properties of DNA upon interaction with dipyrandium.

Interaction of dipyrandium with DNA and its dependence on the base sequence was studied using circular dichroism. It was found that calf thymus DNA and polynucleotide duplexes with alternating purine-pyrimidine sequences containing GC basepairs underwent similar alterations in the chiroptical properties upon binding of dipyrandium. The alterations suggest that these DNAs have similar B-type structures which may kink at the dipyrandium binding sites. On the other hand, poly(dA-dT).poly(dA-dT) and especially poly(dA-dU).poly(dA-dU) exhibit some features of A-type structure. Poly(dA-dT).poly(dA-dT) changes its chiroptical properties little when complexed with dipyrandium, as if it contained some type of kinks as equilibrium structural elements.

Sequence specific damage of DNA induced by reducing sugars.

Reducing sugars induced alkali-labile sites in DNA. The DNA reacted with D-fructose 6-phosphate or D-fructose in the presence of Cu2+ was cleaved by the treatment with aqueous piperidine at 90 degrees C for 30 min. Alkali-labile sites were induced frequently at the pyrimidine residues, especially at the pyrimidine residues in pyrimidine-purine (5'----3') sequences. Transition metal ions such as Cu2+ and oxygen radicals such as hydrogen peroxide were possibly involved in the induction of alkali-labile sites.

Intervening sequences in human fetal globin genes adopt left-handed Z helices.

The large intervening sequences ( IVS2 ) of three human fetal globin genes contain tracts of alternating purine-pyrimidine sequences approximately 40-60 base pairs in length which adopt left-handed Z DNA helices under the influence of negative supercoiling. The amount of negative supercoiling (approximately 0.045) required for the right- to left-handed transitions is within the physiological range. The structural aberrations between the right- and left-handed helices were mapped by sequencing the S1 nuclease cleavage sites. Two-dimensional gel electrophoretic analyses of the supercoil-induced relaxation served to characterize the type and length of left-handed structure. Furthermore, binding studies with several types of antibodies confirmed the presence of left-handed helices. Since these simple sequences appear to be hotspots for recombination and gene conversion, unusual DNA conformations may participate in genetic expression.