Chemical probing of the homopurine.homopyrimidine tract in supercoiled DNA at single-nucleotide resolution

Local structure of the homopurine.homopyrimidine tract in a supercoiled plasmid pEJ4 was studied using chemical probes at single-nucleotide resolution. The conformation of the homopyrimidine strand was probed by osmium tetroxide, pyridine (Os,py) while that of the homopurine strand was tested by diethyl pyrocarbonate (DEPC), i.e. by probes reacting preferentially with single-stranded DNA. At weakly acidic pH values, a strong Os,py attack on three nucleotides at the centre of the (dC-dT)16 block and a weaker attack on two nucleotides at the end of the block were observed. DEPC modified adenines in the 5'-half of the homopurine strand. Os,py modification at the centre of the block corresponded to the loop of the hairpin formed by the homopyrimidine tract, while DEPC modification corresponded to the unstructured half of the homopurine strand in the model of protonated triplex H form of DNA.     

Electron microscopy of supercoiled pEJ4 DNA containing homopurine.homopyrimidine sequences

Supercoiled pEJ4 DNA (a derivative of pUC19 containing an insert with 60-bp-long homopurine.homopyrimidine tract from the sea urchin P. miliaris histone gene spacer) was investigated by electron microscopy using three different spreading techniques i.e., formamide and aqueous variants of the Kleinschmidt technique and protein-free benzyldimethyl-alkyl ammonium chloride (BAC) technique at different pHs. If the specimens for electron microscopy were prepared at pH 5.6 and pH 4.0 (i.e., under conditions where the homopurine.homopyrimidine tract assumes an unusual conformation) a single thick "stem" or a "denaturation bubble" in a large number of DNA molecules were observed. No such changes were found in samples prepared at neutral pH and in linearized pEJ4 DNA prepared at pH 5.6. In specimens of a control supercoiled pUC19 DNA prepared at pH 5.6 and 4.0 practically no local changes were detected. The "denaturation bubbles" were observed by BAC techniques (probably due to secondary local DNA denaturation during the specimen preparation) while the more gentle formamide technique revealed only "stems". The "stems" were almost always positioned at the sites where the curvature of supercoiled DNA molecules occurred. The results are in agreement with presence of a protonated triplex H-form in homopurine.homopyrimidine tract bringing the first evidence of curvature or kinking of the DNA molecule connected with the occurrence of the H-form in supercoiled DNA.     

A novel palindromic triple-stranded structure formed by homopyrimidine dodecamer d-CTTCTCCTCTTC and homopurine hexamer d-GAAGAG.

We have carried out NMR and molecular mechanics studies on a complex formed when a palindromic homopyrimidine dodecamer (d-CTTCTCCTCTTC) and a homopurine hexamer (d-GAAGAG) are mixed in 1:1 molar ratio in aqueous solutions. Such studies unequivocally establish that two strands of each oligomer combine to form a triple-stranded DNA structure with a palindromic symmetry and with six T.A:T and six C+. G:C hydrogen-bonded base triads. The two purine strands are placed head to head, with their 3' ends facing each other in the center of the structure. One-half of each pyrimidine strand contains protonated and the other half contains non-protonated cytosines. The two half segments containing protonated cytosines are hydrogen bonded to each of the two purine hexamers through Hoogsteen T.A and C+.G base pairing. The segments containing non-protonated cytosines are involved in Watson-Crick (A:T and G:C) base pairing. This leads to a palindromic triplex with a C2-dyad symmetry with respect to the center of the structure. The complex is less stable at neutral pH, but the cytosines involved in Hoogsteen base pairing remain protonated even under these conditions. Molecular mechanics calculations using NMR constraints have provided a detailed three-dimensional structure of the complex. The entire stretches of purine, and the pyrimidine nucleotides have a conformation close to B-DNA.     

Structural polymorphism exhibited by a homopurine.homopyrimidine sequence found at the right end of human c-jun protooncogene

Homopurine·homopyrimidine (Pu·Py) tracts are likely to play important biological role in eukaryotes. Using circular dichroism, UV-thermal denaturation and gel electrophoresis, we have analyzed the structural polymorphism of a 21-bp Pu·Py DNA segment within human c-jun protooncogene 3′-region, a potential target for triplex formation. Results show that below physiological pH and in the presence of Na⁺/K⁺ with Mg²⁺ the duplex is destabilized/disproportionated, resulting in strand mediated structural transitions to the self-associated structures of G- and C-rich strands separately, identified as G-quadruplex and i-motif species. A significant differential behavior of the monovalent cations was observed, accordingly the presence of Na⁺ in acidic as well as neutral pH facilitated the duplex formation, while K⁺ favored the formation of self-associated structures. In Na⁺ and Mg²⁺, under acidic and neutral pH conditions, the duplex displayed triphasic and biphasic melting profiles, respectively. This self-association property of oligonucleotides might limit their use as duplex targets in triplex formation. Study is also relevant for understanding structural and biological properties of DNA sequence containing homopurine tracts.     

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.     

Identification of a long stretch of homopurine.homopyrimidine sequence in a cluster of retroposons in the human genome

A cluster of nine retroposons of four different types in a 6221 base EcoRI DNA fragment was isolated from a human fetal liver genomic library using a human nucleophosmin (B23) cDNA as a probe. These retroposons are: (1) a solitary HERV-K long terminal repeat upstream from; (2) a nucleophosmin processed pseudogene; (3) six Alu repeated sequences interspersed in both directions; and (4) a truncated Kpn repeated sequence integrated by an Alu monomer and the HERV-K long terminal repeat. Sequence analysis shows that the nucleophosmin pseudogene contains a long stretch (135 base-pairs) of homopurine.homopyrimidine (Pur.Pyr) sequence. S1 and P1 nuclease digestion indicated that this sequence was able to adopt a non-B-DNA triplex structure under either acidic or neutral conditions. This finding is the first example of the association of a potential DNA triplex structure with a cluster of retroposons.     

Imaging and nanodissection of individual supercoiled plasmids by atomic force microscopy.

The atomic force microscope (AFM) was used to image supercoiled plasmid DNA deposited on a mica surface in either a hydrated or desiccated state. Hydrated plasmid was precisely cut by the scanning tip at a location determined by the instrument operator. Small pieces of DNA (100-150 nm in length) were excised and deposited adjacent to the dissected plasmid, demonstrating that it is possible to remove and manipulate genomic DNA fragments, unresolvable by light microscopy, from defined chromosomal locations by AFM.     

The interactions of enzyme and chemical probes with inverted repeats in supercoiled DNA

In negatively supercoiled DNA molecules some inverted repeat sequences adopt a perturbed conformation which is characterised by the following properties. They are centrally hypersensitive to single-strand-specific nucleases such as S1, and to a much lower extent the flanking regions may also be sensitive. They are also hypersensitive to modification by bromoacetaldehyde, particularly in their flanking region. They may be resistant to endonucleolysis by restriction enzymes and are cleaved (resolved) by a T4 resolving enzyme. All these properties can only be consistently explained by a model in which the inverted repeat adopts a cruciform structure. This property has been shown to depend sharply on a superhelix density, and the transition to nuclease sensitivity is accompanied by a marked alteration in the overall molecular geometry as judged by frictional properties. The probable dynamics of these structures are discussed.     

Dynamics in the isomerization of intramolecular DNA triplexes in supercoiled plasmids.

We report here kinetic and thermodynamic studies on differential isomerization of intramolecular Pyr*Pur.Pyr triplexes in supercoiled plasmids. Two structural isomers of the triplex exist: one with the 3'-half of the Pyr strand as the third strand (H-y3 form) and the other with the 5'-half as the third strand (H-y5 form). The relative populations of the two triplex isomers was determined using the chemical probe with diethyl pryrocarbonate as a function of incubation time. The results demonstrated that triplexes were formed rapidly after a pH change from pH 8.0 to 5.0 and that the initial population of the two isomers exponentially changed with incubation time to reach true thermodynamic equilibrium with a time constant of 0.6-10 h, depending on temperature and the presence of Mg2+. The results clearly demonstrated that interconversion occurs between the two isomers and that the presence of Mg2+ generally retarded the interconversion rates. Kinetic and thermodynamic analyses of the relative populations of the two isomers revealed that the apparent energy barrier for transition from duplex to the H-y3 form is higher than that to the H-y5 form, but H-y3 is more stable in enthalpy terms than H-y5. Therefore, H-y3 is kinetically inferior but thermodynamically favored at higher supercoil levels in plasmids. The presence of Mg2+ resulted in both a kinetic and a thermodynamic preference for H-y5 formation, relative to the H-y3 form.     

A comparison of the circular dichroism spectra of synthetic DNA sequences of the homopurine . homopyrimidine and mixed purine- pyrimidine types.

We have obtained the ultraviolet circular dichroism spectra of two repeating trinucleotide DNAs, poly [d(A-G-G).d(C-C-T)] and poly[d(A-A-G).d(C-T-T)], that have all purines on one strand and all pyrimidines on the other. These spectra, together with spectra of other synthetic polymers, can be combined to give 3 first-neighbor calculations of the spectrum of poly[d(A).d(T)] and 2 first-neighbor calculations of the spectrum of poly [d(G).d(C)]. The results show (1) that first-neighbor calculations utilizing only spectra of homopurine.homopyrimidine DNA sequences are no more accurate than are similar calculations that involve spectra of mixed purine-pyrimidine sequences, demonstrating that double-stranded homopurine.homopyrimidine sequences do not obviously belong to a special class of secondary conformations, and (2) that the wavelength region above 250 nm in the CD spectra of synthetic DNAs is least predictable from first-neighbor equations, probably because this region is especially sensitive to sequence-dependent conformational differences.     

Osmium tetroxide probing of local DNA structure in linear and supercoiled plasmids containing curvature-inducing sequences

Recombinant plasmids pK1A108, pK3A108, pK4A108 and pK5/6T217 containing 80 +/- 1 base pair inserts with different curvature-inducing sequences were studied using the DNA structure probe osmium tetroxide in the presence of pyridine (Os, py). The insertion sequences of the plasmids pK1A108, pK3A108, and pK4A108 are strongly related while the degree of curvature increases from pK1A108 (no curvature) less than pK3A108 less than pK4A108 less than pK5/6T217. The Os, py probe reacts selectively with single-stranded and distorted double-stranded regions in the DNA double helix. Nuclease S1 was used to recognize and cleave regions made permanently single-stranded due to osmium recognize and cleave regions made permanently single-stranded due to osmium modification. In linearized plasmids treatment with Os, py produced no S1-detectable site-specific modification. This result is in agreement with models suggested for DNA curvature; in general, continuous base pairing and base stacking is considered through different sequence blocks as well as through structural junctions. Os, py-probing of the plasmids in the supercoiled state also resulted in no S1-detectable site-specific modification within the inserts of pK1A108, pK3A108, and pK4A108 plasmids (while the regions containing inverted repeat nucleotide sequences in these plasmids were site-specifically modified). In contrast, supercoiled pK5/6T217 DNA was site-specifically modified within the curvature-inducing insert sequence. The nucleotide sequence of the insert of this plasmid strongly differs from the insertion sequences of the other three plasmids; it is extremely AT-rich and contains regularly arranged dAGAGA and dATATA sequences. The structural distortion observed in supercoiled pK5/6T217 is most probably due to the presence of these sequences in a particular arrangement in the insertion sequence.     

Analytical and biological characterization of supercoiled plasmids purified by various chromatographic techniques

Supercoiled plasmids are an important component of gene-based delivery vehicles. A number of production methods for clinical applications have been developed, each resulting in very high-quality product with low levels of residual contaminants. There is, however, no consensus on the optimal methods to characterize plasmid quality, and further, to determine if these methods are predictive of either product stability or biological activity. We have produced two plasmids using four production purification methodologies based on PolyFlo and hydrophobic interaction chromatography (HIC), either alone or in tandem processes. In each case, the product was analyzed using standard molecular biological methods. We also performed a number of biophysical analyses such as dynamic light scattering (DLS), circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). Minimal differences were detected among the preparations based on the more standard molecular biological methods. Some small differences were detected, however, using biophysical techniques, particularly FTIR and DSC, which may reflect small variations in plasmid tertiary structure and thermal stability. Stability after heat exposure at 60 degrees C, exposure to fetal bovine serum and long-term storage at 4 degrees C varied between plasmids. One plasmid showed no difference in stability depending on the production process, but the other showed significant differences. Evaluation in vivo in models for gene immunization and gene therapy showed significant differences in the response depending on the method of purification. Preparations using a tandem process of PolyFlo used in two separation modes provided higher biological activity compared to a tandem HIC/PolyFlo process or either resin used alone in a single column process. These data indicate that the process by which supercoiled plasmids are made can influence plasmid stability and biological activity and emphasize the need for more rigorous methods to evaluate supercoiled plasmids as gene-delivery vehicles.     

Structural interconversion of alternating purine-pyrimidine inverted repeats cloned in supercoiled plasmids.

Two self complementary oligonucleotides, T(GC)4AT(GC)4ACATG and C(GC)2(AT)5 (GC)3ATG, were synthesized and cloned into plasmids. Negative supercoiling causes a structural transition in the primary helix of both inserts. The first sequence converts into the left-handed helix, whereas the second sequence undergoes a transition into a cruciform or a Z-type structure depending on the experimental conditions employed. This has been deduced from the mapping of S1 nuclease sensitive sites, OsO4-sensitive sites, DEP modification pattern and relaxation studies. In addition, the differential effect of 5-cytosine methylation and binding of the AT-specific drug distamycin on these transitions further supports this interpretation. Thus, it is demonstrated, that the same sequence which is both inverted repeat and alternating purine-pyrimidine type may adopt either the left-handed conformation or the cruciform structure in response to the superhelical stress. Formation of the Z-type helix can be transmitted through the d(AT)n region which is 10 bp in length.     

The identification of nuclear proteins that bind the homopyrimidine strand of d(GA.TC)n DNA sequences, but not the homopurine strand.

Alternating d(GA.TC)(n)DNA sequences, which are abundant in eukaryotic genomes, can form altered DNA structures. Depending on the environmental conditions, the formation of (GA.GA) hairpins or [C+T(GA.TC)] and [GA(GA.TC)] intramolecular triplexes was observed in vitro. In vivo, the formation of these non-B-DNA structures would likely require the contribution of specific stabilizing factors. Here, we show that Friend's nuclear extracts are rich in proteins which bind the pyrimidine d(TC)(n)strand but not the purine d(GA)n strand (NOGA proteins). Upon chromatographic fractionation, four major proteins were detected (NOGA1-4) that have been purified and characterized. Purified NOGAs bind single-stranded d(TC)n with high affinity and specificity, showing no significant affinity for either d(GA)n or d(GA.TC)nDNA sequences. We also show that NOGA1, -2 and -3, which constitute the three most abundant and specific NOGA proteins, correspond to the single-stranded nucleic acid binding proteins hnRNP-L, -K and -I, respectively. These results are discussed in the context of the possible contribution of the NOGA proteins to the stabilization of the (GA.GA) and [GA(GA.TC)] conformers of the d(GA.TC)n DNA sequences.     

Intramolecular DNA triplexes in supercoiled plasmids. I. Effect of loop size on formation and stability

The capacity of four oligopurine.oligopyrimidine (pur.pyr) sequences with different lengths of interruptions in the center [GAA)4(N)n(GAA)4G) (n = 3, 5, 7, and 9) to adopt intramolecular DNA triplexes was evaluated in recombinant plasmids. The hyperreactive patterns of the pur.pyr inserts to specific chemical probes (OsO4, diethyl pyrocarbonate, and dimethyl sulfate) at the base pair level demonstrate that intramolecular triplexes with identical 12-base triads in the stem but with different loop sizes (4, 6, 8, and 10 bases) can form in supercoiled plasmids. Furthermore, the extent of OsO4 modification was measured as a function of temperature and of average negative supercoil density. In addition, the transition free energy of B-DNA to triplexes at pH 4.5 was determined by two-dimensional electrophoresis. These comparative studies show that longer loops require more supercoil energy for triplex formation and are less thermostable than triplexes with shorter loops. Also, it may be that not only the loop size but the base composition of the loop region affects the structural transition and triplex stability. Thus, these results significantly broaden the range of natural pur.pyr sequences that may adopt triplexes.     

An assessment of the Z-DNA forming potential of alternating dA-dT stretches in supercoiled plasmids

The ability of a stretch of alternating dA-dT to adopt the left-handed Z form has been assessed by examining the behavior of the sequence d(CG)6(TA)4(CG)6 contained in the plasmid pBR322. The structural transition occurring within this sequence as a function of negative superhelicity was analyzed by several methods, including (1) the supercoiling-dependent unwinding of the insert as determined by two-dimensional gel electrophoresis, (2) the binding of anti-Z-DNA antibodies to the insert, (3) the sensitivity of the sequence to a single strand specific endonuclease, and (4) the sensitivity of the insert to digestion by a restriction endonuclease that cuts within the d(CG)6 segments when in the right-handed form. These studies have shown that in negatively supercoiled DNA the two d(CG)6 portions of the insert adopt the Z form, while the central d(TA)4 segment forms an underwound structure with a helical repeat that is best approximated as being intermediate between the B form and the Z form. A statistical mechanical treatment of the unwinding of the insert as a function of negative superhelicity provides an estimate of the minimum free energy required to convert an A-T bp from the B form to the Z form, as well as the free energy associated with the conversion of an A-T bp from the B form to the unwound form. These results strongly indicate that Z DNA is an unfavored structural alternative for stretches of d(AT)n in negatively supercoiled DNA.