DNA structural transitions within the PKD1 gene.

Autosomal dominant polycystic kidney disease (ADPKD) affects over 500 000 Americans. Eighty-five percent of these patients have mutations in the PKD1 gene. The focal nature of cyst formation has recently been attributed to innate instability in the PKD1 gene. Intron 21 of this gene contains the largest polypurine. polypyrimidine tract (2.5 kb) identified to date in the human genome. Polypurine.polypyrimidine mirror repeats form intramolecular triplexes, which may predispose the gene to mutagenesis. A recombinant plasmid containing the entire PKD1 intron 21 was analyzed by two-dimensional gel electrophoresis and it exhibited sharp structural transitions under conditions of negative supercoiling and acidic pH. The superhelical density at which the transition occurred was linearly related to pH, consistent with formation of protonated DNA structures. P1 nuclease mapping studies of a plasmid containing the entire intron 21 identified four single-stranded regions where structural transitions occurred at low superhelical densities. Two-dimensional gel electrophoresis and chemical modification studies of the plasmid containing a 46 bp mirror repeat from one of the four regions demonstrated the formation of an H-y3 triplex structure. In summary, these experiments demonstrate that a 2500 bp polypurine.polypyrimidine tract within the PKD1 gene is capable of forming multiple non-B-DNA structures.     

Unpaired structures in SCA10 (ATTCT)n.(AGAAT)n repeats

A number of human hereditary diseases have been associated with the instability of DNA repeats in the genome. Recently, spinocerebellar ataxia type 10 has been associated with expansion of the pentanucleotide repeat (ATTCT)(n).(AGAAT)(n) from a normal range of ten to 22 to as many as 4500 copies. The structural properties of this repeat cloned in circular plasmids were studied by a variety of methods. Two-dimensional gel electrophoresis and atomic force microscopy detected local DNA unpairing in supercoiled plasmids. Chemical probing analysis indicated that, at moderate superhelical densities, the (ATTCT)(n).(AGAAT)(n) repeat forms an unpaired region, which further extends into adjacent A+T-rich flanking sequences at higher superhelical densities. The superhelical energy required to initiate duplex unpairing is essentially length-independent from eight to 46 repeats. In plasmids containing five repeats, minimal unpairing of (ATTCT)(5).(AGAAT)(5) occurred while 2D gel analysis and chemical probing indicate greater unpairing in A+T-rich sequences in other regions of the plasmid. The observed experimental results are consistent with a statistical mechanical, computational analysis of these supercoiled plasmids. For plasmids containing 29 repeats, which is just above the normal human size range, flanked by an A+T-rich sequence, atomic force microscopy detected the formation of a locally condensed structure at high superhelical densities. However, even at high superhelical densities, DNA strands within the presumably compact A+T-rich region were accessible to small chemicals and oligonucleotide hybridization. Thus, DNA strands in this "collapsed structure" remain unpaired and accessible for interaction with other molecules. The unpaired DNA structure functioned as an aberrant replication origin, in that it supported complete plasmid replication in a HeLa cell extract. A model is proposed in which unscheduled or aberrant DNA replication is a critical step in the expansion mutation.     

Non-B right-handed DNA conformations of homopurine.homopyrimidine sequences in the murine immunoglobulin C alpha switch region

The switch region of IgA immunoglobulin in mice cloned into a recombinant plasmid contains a supercoil-dependent S1 nuclease hypersensitive site, indicative of a non-B-DNA secondary structure. This site maps to the (AGGAG)28 direct repeat (DR2) of the alpha switch region and appears at a negative superhelical density of greater than 0.02. Studies with P1 nuclease and bromoacetaldehyde indicate that this structure is also present at neutral pH. S1 nuclease sensitivity is retained for the shorter repeat (AGGAG)6GA in a recombinant plasmid but is not seen for the repeat (CTGAG)6, corresponding to the DR1 repeat of the alpha switch region, or in a sequence corresponding to a portion of the consensus sequence which contains a short stretch of alternating pyrine-pyrimidine residues. Fine mapping of the (AGGAG)6GA and flanking sequences with dimethyl sulfate, bromoacetaldehyde, osmium tetroxide, and diethyl pyrocarbonate reveals an asymmetric pattern of modification dependent on both pH and supercoiling. Two-dimensional gel electrophoresis at low pH shows the relaxation of 3 superhelical turns on formation of this structure by the (AGGAG)6GA repeat. These results are most consistent with the formation of an intramolecular triple-strand.     

Cruciform extrusion in plasmids bearing the replicative intermediate configuration of a poxvirus telomere

The transition from lineform DNA to cruciform DNA (cruciformation) within the cloned telomere sequences of the Leporipoxvirus Shope fibroma virus (SFV) has been studied. The viral telomere sequences have been cloned in recombination-deficient Escherichia coli as a 322 base-pair, imperfect palindromic insert in pUC13. The inverted repeat configuration is equivalent to the arrangement of the telomere structures observed within viral DNA replicative intermediates. A major cruciform structure in the purified recombinant plasmid has been identified and mapped using, as probes, the enzymes AflII, nuclease S1 and bacteriophage T7 endonuclease I. It was extruded from the central axis of the cloned viral inverted repeat and, by unrestricted branch migration, attained a size commensurate with the superhelical density of the plasmid molecule at native superhelical densities. This major cruciform extrusion event was the only detectable duplex DNA perturbation, induced by negative superhelical torsion, in the insert viral sequences. No significant steady-state pool of extruded cruciform was identified in E. coli. However, the identification of a major deletion variant generated even in the recombination-deficient E. coli strain DB1256 (recA recBC sbcB) suggested that the cruciform may be extruded transiently in vivo. The lineform to cruciform transition has been further characterized in vitro using two-dimensional agarose gel electrophoresis. The transition was marked by a high energy of formation (delta Gf = 44 kcal/mol), and an apparently low activation energy that enabled facile transitions at physiological temperatures provided there was sufficient torsional energy. By comparing cruciformation in a series of related bidirectional central axis deletions of the telomeric insert, it has been concluded that the presence of extrahelical bases in the terminal hairpin structures contributes substantially to the high delta Gf value. Also, viral sequences flanking the extruded cruciform were shown to influence the measured delta Gf value. Several general features of poxvirus telomere structure that would be expected to influence the facility of cruciform extrusion are discussed along with the implications of the observed cruciform transition event on the replicative process of poxviruses in vivo.     

The segment inversion site of herpes simplex virus type 1 adopts a novel DNA structure

The 12-base pair (bp) tandem direct repeat sequences (DR2) at the joint region (a sequence) of herpes simplex virus type 1 (strain F) adopt a new type of DNA conformation under the influence of negative supercoiling. The novel conformation is dependent on the number of the DR2 repeats; the 19 mer (228 bp total) and the 14 mer (168 bp) readily form the alternate structure whereas pentamer, trimer, and dimer repeats show somewhat different properties. S1 and P1 nuclease studies reveal that the new conformation has a major structural aberration at its center and conformational periodicities which are not identical on the complementary strands. Also, the effect of salt and pH, the location of reaction with bromo- and chloroacetaldehyde, the type of sequence (direct repeat) involved, and the nature and extent of supercoil-induced relaxations demonstrate that this structure differs from previously recognized conformations including left-handed Z helices, cruciforms, bent DNA, and slipped structures. We propose the existence of a novel conformation, anisomorphic DNA, with different structures on the complementary strands which elicit a structural aberration at the physical center of the tandem sequences. Since the oligopurine X oligopyrimidine sequence may be inherently inflexible, this supercoil-induced structural change and the physical stress on these inserts in recombinant plasmids tend to deform (crack) the DR2 sequences at their centers. Possible roles for anisomorphic DNA in the functions of this segment of intense biological activity are proposed.     

The role of DNA sequence in the formation of Z-DNA versus cruciforms in plasmids

The capacities of four synthetic sequences containing runs of perfectly alternating purine-pyrimidine base pairs (bp) to adopt left-handed structures were evaluated in a homologous family of recombinant plasmids. All the sequences had the same G+C content (50%) and consisted of simple tetranucleotide repeat units but differed in the relative orientations of these units. For some of the sequences, several alternate secondary structures were theoretically possible; a variety of probes (S1 nuclease, bromoacetaldehyde, OsO4, T7 gene 3 endonuclease, supercoil-induced gel relaxation studies) under a wide range of reaction conditions was used to determine which structures were adopted as a function of superhelical stress. The precise positions at the bp level of reactions with these chemical and enzymatic probes were determined. We conclude that for short (20-24 bp) sequences containing runs of alternating (T-G) and (C-A), the cruciform state is preferred over the similarly allowable left-handed form provided that symmetry constraints allow. However, these sequences can be induced to form a left-handed helix under appropriate conditions. This is the first demonstration of plasmid inserts which will adopt more than one unusual DNA structure in response to negative superhelical stress. The structural properties of a molecule containing a Z-Z junction were studied, and we conclude that the disruption caused by this feature extends over only a few bp although it requires a high energetic penalty.     

Formation of intramolecular triplex in homopurine-homopyrimidine mirror repeats with point substitutions.

We have used two-dimensional gel electrophoresis to study the structural transition to the triplex H form of sequences 5'-AAGGGAGAAXGGGGTATAGGGGYAAGAGGGAA-3' where X and Y are any DNA bases. The transition was observed at acid pH under superhelical stress. For X = Y = A or X = Y = G the sequences corresponded to homopurine-homopyrimidine mirror repeats (H-palindrome) which are known to adopt the H form under acid pH and superhelical stress. We have shown that the H form is actually formed for all X and Y, though in cases other than X = Y = A and X = Y = G the transition requires larger negative superhelical stress. Different substitutions require different superhelicity levels for the transition to occur. Theoretical analysis of the data obtained made it possible to estimate the energy cost of triplex formation due to all possible mismatched base triads.     

Supercoil-induced Z-DNA formation within 5'-end of chicken myb proto-oncogene

We have analyzed the recently sequenced and characterized 2.9 kb fragment derived from the 5'-end of chicken myb proto-oncogene with respect to structural perturbations induced by DNA supercoiling. Within the first intron a 50 bp sequence stretch was localized, starting approximately 450 nucleotides downstream from putative ATG initiation codon, which forms a non-B-DNA structure. Fine mapping with structural probes revealed the three adjacent regions with imperfect purine-pyrimidine alternation creating together relatively long Z-forming tract, parts of which may undergo a B-Z DNA transition at different superhelical densities.     

Zintrons in rat alpha-lactalbumin gene

The eukaryotic genome is characterised by the occurrence of a large amount of repetitive DNA which exists within and around coding sequences. Random repeats of (TG)n sequences have been observed in several introns. In this paper we show that (TG)14 and (TG)24 sequences present in the third intron of rat alpha-lactalbumin gene adopt left-handed Z-helices under varying superhelical densities. The overall sequence of the parent plasmid further influenced the level of supercoiling at which the B to Z transition could be induced in (TG)n sequences. Such Z-potential intervening sequences (zintrons) could act as buffers to maintain desired levels of supercoiling near the transcribed sequences.     

The influence of tertiary structural restraints on conformational transitions in superhelical DNA.

This paper examines theoretically the effects that restraints on the tertiary structure of a superhelical DNA domain exert on the energetics of linking and the onset of conformational transitions. The most important tertiary constraint arises from the nucleosomal winding of genomic DNA in vivo. Conformational transitions are shown to occur at equilibrium at less extreme superhelicities in DNA whose tertiary structure is restrained than in unrestrained molecules where the residual linking difference alpha res (that part of the superhelical deformation which is not absorbed by transitions) may be freely partitioned between twisting and bending. In the extreme case of a rigidly held tertiary structure, this analysis predicts that the B-Z transition will occur at roughly half the superhelix density needed to drive the same transition in solution, other factors remaining fixed. This suggests that superhelical transitions may occur at more moderate superhelical deformations in vivo than in solution. The influence on transition behavior of the tertiary structural restraints imposed by gel conditions also are discussed.     

Relationship between superhelical density and cruciform formation in plasmid pVH51

The relative stability of the cruciform state at the large inverted repeat of plasmid pVH51 is measured. At physiological superhelical densities, the cruciform state is present in a high percentage of the plasmid molecules. Investigation of the relationship between negative superhelical density and cruciform prevalence reveals a sharp transition from an undetectable level to a relatively stable state. This transition occurs over the negative superhelical density range of 0.046 to 0.066. Estimates of the free energy contribution to cruciform formation resulting from loss of negative superhelical turns suggest that about 22 kcal/mol are required to generate the cruciform structure at this site in pVH51.     

Energetics of the strand separation transition in superhelical DNA.

In this paper the values of three free energy parameters governing the superhelical strand separation transition are determined by analysis of available experimental data. These are the free energy, a, needed to initiate a run of separation, the torsional stiffness, C, associated with interstrand winding of the two single strands comprising a separated site and the coefficient, K, of the quadratic free energy associated to residual linking. The experimental data used in this analysis are the locations and relative amounts of strand separation occurring in the pBR322 DNA molecule and the measured residual linking, both evaluated over a range of negative linking differences. The analytic method used treats strand separation as a heteropolymeric, co-operative, two-state transition to a torsionally deformable alternative conformation, which takes place in a circular DNA molecule constrained by the constancy of its linking number. The values determined for these parameters under the experimental conditions (T = 310 K, pH = 7.0, monovalent cation concentration = 0.01 M) are a = 10.84(+/- 0.2) kcal/mol, C = 2.5(+/- 0.3) x 10(-13) erg/rad2 and K = 2350(+/- 80) RT/N, where N is the molecular length in base-pairs. In order to assess the accuracy of the author's theoretical methods, these free energy parameters are incorporated into the analysis of superhelical strand separation in different molecules and under other conditions than those used in their evaluation. First, the temperature dependence of transition is treated, then superhelical strand separation is analyzed in a series of DNA molecules having systematic sequence modifications, and the results of these theoretical analyses are compared with those from experiments. In all molecules, transition is predicted in the range of linking differences where it is seen experimentally. Moreover, it occurs at the specific sequence locations that the analysis predicts, and with approximately the predicted relative amounts of transition at each location. The known sensitivities of this transition to changes of temperature and to small sequence modifications are predicted in a quantitatively precise manner by the theoretical results. The demonstrated high-level precision of these theoretical methods provides a tool for the screening of DNA sequences for sites susceptible to superhelical strand separation, some of which may have regulatory or other biological significance.     

Nuclease S1-sensitive sites on superhelical DNA molecules carrying the LTR region of Moloney murine leukemia virus

The long terminal repeat (LTR) from proviral DNA of Moloney murine leukemia virus (Mo-MLV) was cloned on a derivative of pBR322, and after introducing superhelical torsions into the resulting recombinant, the sites of conformational transition were investigated by the nuclease S1-digestion method. With an increase in the negative linking differences, fourteen dominant cutting sites were identified, of which two were mapped inside the LTR and one at the 3' end of the LTR. By searching the sequence data, all these sites were localized in the regions having either palindromic sequences or AT-rich sequences. Free energy calculation for the local secondary structure on one strand indicated that nuclease S1 attacked the palindromic sequence regions which could form relatively stable hairpin structures. Under the conditions used, no correlation was found between the S1-sensitive sites and the potential Z-DNA-forming regions, including those within the enhancer sequence.     

The effect of zinc on the secondary structure of d(GA.TC)n DNA sequences of different length: a model for the formation *H-DNA.

Alternating d(GA.TC)n DNA sequences are known to undergo transition to *H-DNA in the presence of zinc. Here, the effect of zinc on the secondary DNA structure of d(GA.TC)n sequences of different length (n = 5, 8, 10 and 19) was determined. Short d(GA.TC)n sequences form *H-DNA with a higher difficulty than longer ones. At bacterial negative superhelical density (- sigma = 0.05), zinc still induces transition to the *H-DNA conformation at a d(GA.TC)10 sequence but shorter sequences do not form *H-DNA. Transition to *H-DNA at a d(GA.TC)8 sequence is observed under conditions which destabilize the DNA double helix such as high negative supercoiling or low ionic strength. Our results indicate that a first step in the transition to *H-DNA is the formation of a denaturation bubble at the centre of the repeated DNA sequence, suggesting that the primary role of zinc is to induce a local denaturation of the DNA double helix. Subsequently, zinc might also participate in the stabilization of the altered DNA conformation through its direct interaction with the bases. Based on these results a model for the formation of *H-DNA is proposed.     

Transition of a cloned d(AT)n-d(AT)n tract to a cruciform in vivo.

A 34 base pair tract of the simple repeating dinucleotide d(AT)n-d(AT)n cloned into a 2.4 kb polylinker plasmid vector undergoes a structural transition in response to negative superhelical coiling. The transition has been characterized by 2 dimensional gel electrophoresis, mapping of S1, P1 and T7 endonuclease 1 sensitive sites, and mapping of sites that are sensitive to modification by bromoacetaldehyde. After S1 nuclease treatment it is possible to trap supercoiled species that are nicked on one or both strands near the center of the palindrome. These data show that the alternate state adopted by the d(AT)n-dAT)n insert is a cruciform rather than a Z conformation. Unlike other B-cruciform transitions the transition in d(AT)n-d(AT)n has a low activation energy and the transition is facilitated by the presence of magnesium ions. Evidence from in-vivo topoisomer distributions is presented which shows that under conditions of blocked protein synthesis the d(AT)n-d(AT)n insert will spontaneously adopt the cruciform state in-vivo in E. coli.     

A pH-dependent structural transition in the homopurine-homopyrimidine tract in superhelical DNA

We have inserted the 509-bp-long fragment of sea urchin P. miliaris histone gene spacer region into plasmid pUC19. The fragment contains the 60-bp-long homopurine-homopyrimidine tract that is known to be hypersensitive to the S1 endonuclease. Using two-dimensional gel electrophoresis we have observed a sharp structural transition in the insert with increasing DNA superhelicity. As in the cases of cruciform and Z form formation, the observed transition partly relaxes the superhelical stress. In contrast with the other two well documented transitions, the observed transition strongly depends on pH. At pH7 and above the transition occurs at negative superhelicities exceeding the physiological range (- sigma greater than 0.08). For pH6 the transition occurs at -sigma = 0.055, whereas for pH4.3 it takes place at -sigma = 0.001. A comprehensive analysis of the obtained data has made it possible to define the nature of the observed transition. We conclude that under superhelical stress or/and at low pH homopurinehomopyrimidine tracts adopt a novel spatial structure called the H form.