Photochemical probing of the B--a conformational transition in a linearized pUC19 DNA and its polylinker region

We induced the B-to-A conformational transition by ethanol in a linearized pUC19 DNA. A primer extension method was used in combination with UV light irradiation to follow the transition, based on pausing of DNA synthesis due to the presence of damaged bases in the template. Primer extension data highly correlated with the results of another method monitoring the B-A transition, i.e. inhibition of restriction endonuclease cleavage of UV light-irradiated DNA. Primer extension enabled us to locate damaged nucleotides within the region of interest. Most damaged nucleotides were located in B-form trimers, exclusively containing both pyrimidine bases (TTC, TCT, CTC, and CTT), and in a cytosine tetramer. The amount of damaged bases decreased in the course of B-A transition. Some of the damage even disappeared in the A-form, which mainly concerns the C(4) and C(3) blocks. The cleavage was nearly restored in the A-form within this region (Eco88I). On the contrary the decrease of damage was less significant with thymine dimers, only dropping to 50-60% of the B-form level. Consequently, the cleavage with EcoRI and HindIII remained mostly as before the transition (75% and 60% of uncleaved DNA preserved). We found significant differences in the B- and A-form pattern of UV light-damaged bases within the same region (polylinker) of DNA embedded within long (plasmid) or short (127 bp fragment) DNA molecules. The B-A transition of the fragment was found less cooperative than with linearized plasmid, which was confirmed by both CD spectroscopy and restriction cleavage inhibition.     

The flexibility of A-form DNA.

We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350-900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than for B-form DNA. There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2-2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.     

Characterization of a lipophilic plasmid DNA condensate formed with a cationic peptide fatty acid conjugate

In the presence of cationic ligands, DNA molecules can become aggregated into larger particles in a process known as condensation. DNA condensates are of interest as models for the dense packing found in naturally occurring structures such as phage heads and chromatin. They have found extensive application in DNA transfection and also provide convenient models with which to study DNA damage by the direct effect of ionizing radiation. Further, conjugates of cationic peptides with fatty acids may represent a class of attractive ligands for these areas because of their simple synthesis. When plasmid pUC18 is used as the DNA target and N-caproyl-penta-arginine amide (Cap-R(5)-NH(2)) is used as the ligand, the physical properties of the resulting mixtures were characterized using static and dynamic light scattering, sedimentation, dye exclusion, circular dichroism, nanoparticle tracking, and atomic force microscopy. Their chemical properties were assayed using solvent extraction and protection against hydroxyl radical attack and nuclease digestion. Titration of the plasmid with the Cap-R(5)-NH(2) ligand produced sharply defined changes in both chemical and physical properties, which was associated with the formation of condensed DNA particles in the 100-2000 nm size range. The caproyl group at the ligand's N-terminus produced a large increase in the partitioning of the resulting condensate from water into chloroform and in its binding to the neutral detergent Pluronic F-127. Both the physical and chemical data were all consistent with condensation of the plasmid by the ligand where the presence in the ligand of the caproyl group conferred an extensive lipophilic character upon the condensate.     

Poly(dG)-poly(dC) DNA appears shorter than poly(dA)-poly(dT) and possibly adopts an A-related conformation on a mica surface under ambient conditions

Three types of DNA: approximately 2700 bp polydeoxyguanylic olydeoxycytidylic acid [poly(dG)-poly(dC)], approximately 2700 bp polydeoxyadenylic polydeoxythymidylic acid [poly(dA)-poly(dT)] and 2686 bp linear plasmid pUC19 were deposited on a mica surface and imaged by atomic force microscopy. Contour length measurements show that the average length of poly(dG)-poly(dC) is approximately 30% shorter than that of poly(dA)-poly(dT) and the plasmid. This led us to suggest that individual poly(dG)-poly(dC) molecules are immobilized on mica under ambient conditions in a form which is likely related to the A-form of DNA in contrast to poly(dA)-poly(dT) and random sequence DNA which are immobilized in a form that is related to the DNA B-form.     

The Flexibility of A-Form DNA

Abstract

We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350–900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than for B-form DNA. There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2–2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.     

Secondary structure of condensed DNA. wide-angle, small-angle x-ray scattering and circular dichroism

Ethanol precipitated DNA shows a CD spectrum of the +psi-type which is similar to that of DNA in the A-form. DNA condensed with cetyl-trimethylammonium-bromide shows, depending on the condensation velocity, a CD spectrum of the -psi-type, or a CD spectrum only slightly modified from that of DNA in solution. The first spectrum is similar to that of DNA in the C-form, and the second one, to that of DNA in the B-form. Using large-angle X-ray scattering of the three DNA condensates and comparing them with the scattering curves calculated from the atom coordinates for the A-, B-, and C-form of DNA it is shown that the secondary structure of the DNA belongs in all three cases to the B-family. It follows from this result that the secondary structure of DNA alone does not determine the type of CD spectrum. The CD spectrum of condensed DNA is essentially determined by the supramolecular structures of the partially crystalline DNA condensates. These supramolecular structures can be demonstrated by the small-angle X-ray diagrams. The condensation of DNA by ethanol and cetyl-trimethylammonium-bromide proceeds in the form of a partial crystallization of the DNA.     

Condensation of DNA by multivalent cations: experimental studies of condensation kinetics

DNA in viruses and cells exists in highly condensed, tightly packaged states. We have undertaken an in vitro study of the kinetics of DNA condensation by the trivalent cation hexaammine cobalt (III) with the aim of formulating a quantitative, mechanistic model of the condensation process. Experimental approaches included total intensity and dynamic light scattering, electron microscopy, and differential sedimentation. We determined the average degree of condensation, the distribution of condensate sizes, and the fraction of uncondensed DNA as a function of reaction time for a range of [DNA] and [Co(NH(3))(3+)(6)]. We find the following: (1) DNA condensation occurs only above a critical [Co(NH(3))(3+)(6)] for a given DNA and salt concentration. At the onset of condensation, [Co(NH(3))(3+)(6)]/[DNA-phosphate] is close to the average value of 0.54, which reflects the 89-90% charge neutralization criterion for condensation. (2) The equilibrium weight average hydrodynamic radius of the condensates first decreases, then increases with increasing [Co(NH(3))(3+)(6)] as they undergo a transition from intramolecular (monomolecular) to intermolecular (multimolecular) condensation. However, is insensitive to [DNA]. (3) The uncondensed DNA fraction decays approximately exponentially with time. The equilibrium uncondensed DNA fraction and relaxation time decrease with increasing [Co(NH(3))(3+)(6)] but are insensitive to [DNA]. (4) The condensation rate in its early stages is insensitive to [DNA] but proportional to [Co(NH(3))(3+)(6)](xs) = [Co(NH(3))(3+)(6)] - [Co(NH(3))(3+)(6)](crit). (5) Data for low [DNA] and low [Co(NH(3))(3+)(6)] at early stages of condensation are most reliable for kinetic modeling since under these conditions there is minimal clumping and network formation among separate condensates. A mechanism with initial monomolecular nucleation and subsequent bimolecular association and unimolecular dissociation steps with rate constants that depend on the number of DNA molecules in the condensate, accounts reasonably well for these observations.     

Ethanol-induced structural transitions of DNA on mica

The effect of ethanol on the structure of DNA confined to mica in the presence of Mg2+was examined by varying the ethanol concentration and imaging the DNA by atomic force microscopy. Contour length measurements of the DNA show a transition from all-B-form at 0% ethanol to all-A-form at >25% ethanol. At intermediate ethanol concentrations, contour lengths suggest that individual molecules of air-dried DNA are trapped with mixed compositions of A-form and B-form. The relative composition depends on the ethanol concentration. Fitting the length distributions at intermediate ethanol concentrations to a simple binomial model results in an upper bound estimate for the A-form and B-form domains of approximately 54 bp in the individual molecules. In addition to length changes, the apparent persistence length of DNA decreases with increasing ethanol concentration. At high concentrations of ethanol (>20%), DNA formed several higher order structures, including flower shaped condensates and toroids.     

The Intramolecular Impact to the Sequence Specificity of B→A Transition: Low Energy Conformational Variations in AA/TT and GG/CC Steps

Abstract It is well known, that local B→A transformation in DNA is involved in several biological processes. In vitro B?A transition is sequence-specific. The physical basis of this specificity is not known yet. Here we analyze the effect of intramolecular interactions on the structural behavior of the GG/CC and AA/TT steps. These steps exemplify sequence specific bias to the B- or A-form structure. Optimization of potential energy of the molecular systems composed of an octanucle-otide, neutralized by Na(+) and solvated with TIP3P water molecules in rectangular box with periodic boundary conditions gives the statistically representative sets of low energy structures for GG/CC and AA/TT steps in the middle of the diverse flanking sequences. Permissible 3D variations of GG/CC and AA/TT, and correlation of the relative motion of base pairs in these steps were analyzed. AA/TT step permits high variability for low energy conformers in the B-form DNA and small variability for low energy conformers in the A-form DNA. In contrast GG/CC step permits high variability for low energy conformers in the A-form DNA and small variability for low energy conformers in the B-form DNA. The relative motion of base pairs in GG/CC step is high correlated, while in AA/TT step this correlation is notably less. Atom-atom interactions inside-the-step always favors the B-form and their component - stacking interactions (atomatom interactions between nucleic bases) is crucial for the duplex stabilization. Formation of the A-form for both steps is a result of interactions with the flanking sequences and water-cation environment in the box. The average energy difference between conformations presenting B-form and A-form for the GG/CC step is high, while for the AA/TT step it is rather low. Thus, intramolecular interactions in GG/CC and AA/TT steps affect the possible conformational diversity ("conformational entropy") of the A- and B- type structures of DNA step. This determines the known bias of the A-form DNA depending on the enrichment of sequences with GG/CC. If structural tuning during the process of protein-DNA complex formation lead to the local B→A transformation of DNA, it is largely directed by high conformational diversity of GG/CC step in the A-form. In such a case the presence in the target site of both kinds of examined steps ensures the reversible character of ligand binding.     

B-form and A-form DNA in an electric field

The structures of B-form and A-form DNA are studied in 0–70 and 70–80% ethanol solutions, respectively, in an electric field, using linear dichroism. The limiting reduced linear dichroism data of B-form DNA are chain length dependent in 0% ethanol solution. However, there is no such chain length dependence of the limiting reduced linear dichroism of the A-form. Our reslts also suggest that (1) the transition moments at 260 nm lie within the plaxe of the DNA bases, (2) the two allomorphs (A and B forms) of the long chain DNA in solution in the electric field are like the respective classica forms.     

Transformation of yeast with linearized plasmid DNA. Formation of inverted dimers and recombinant plasmid products

The molecular products of DNA double strand break repair were investigated after transformation of yeast (Saccharomyces cerevisiae) with linearized plasmid DNA. DNA of an autonomous yeast plasmid cleaved to generate free ends lacking homology with the yeast genome, when used in transformation along with sonicated non-homologous carrier DNA, gave rise to transformants with high frequency. Most of these transformants were found to harbor a head-to-head (inverted) dimer of the linearized plasmid. This outcome of transformation contrasts with that observed when the carrier DNA is not present. Transformants occur at a much reduced frequency and harbor either the parent plasmid or a plasmid with deletion at the site of the cleavage. When the linearized plasmid is introduced along with sonicated carrier DNA and a homologous DNA restriction fragment that spans the site of plasmid cleavage, homologous recombination restores the plasmid to its original circular form. Inverted dimer plasmids are not detected. This relationship between homologous recombination and a novel DNA transaction that yields rearrangement could be important to the cell, as the latter could lead to a loss of gene function and lethality.     

Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura(+) transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning.     

From triplex to B-form duplex stabilization: reversal of target selectivity by aminoglycoside dimers

Aminoglycosides have been shown to target A-form nucleic acids. Our work has previously shown that neomycin (and other aminoglycosides) bind and stabilize DNA/RNA triplexes and other A-form nucleic acids. We report herein the unexpected B-form duplex stabilization shown by aminoglycoside dimers (neomycin-neomycin and neomycin-tobramycin). The dimers are highly selective for AT rich duplexes and show high affinity (K(a) approximately 10(8)M(-1)) as determined by isothermal titration calorimetry.     

Mechanical properties of high-G.C content DNA with a-type base-stacking

The sequence of a DNA molecule is known to influence its secondary structure and flexibility. Using a combination of bulk and single-molecule techniques, we measure the structural and mechanical properties of two DNAs which differ in both sequence and base-stacking arrangement in aqueous buffer, as revealed by circular dichroism: one with 50% G·C content and B-form and the other with 70% G·C content and A-form. Atomic force microscopy measurements reveal that the local A-form structure of the high-G·C DNA does not lead to a global contour-length decrease with respect to that of the molecule in B-form although it affects its persistence length. In the presence of force, however, the stiffness of high-G·C content DNA is similar to that of balanced-G·C DNA as magnetic and optical tweezers measured typical values for the persistence length of both DNA substrates. This indicates that sequence-induced local distortions from the B-form are compromised under tension. Finally, high-G·C DNA is significantly harder to stretch than 50%-G·C DNA as manifested by a larger stretch modulus. Our results show that a local, basepair configuration of DNA induced by high-G·C content influences the stretching elasticity of the polymer but that it does not affect the global, double-helix arrangement.     

Star-shaped poly(ethylene glycol)-block-polyethylenimine copolymers enhance DNA condensation of low molecular weight polyethylenimines

Star-shaped poly(ethylene glycol)-block-polyethylenimine [star-(PEG-b-PEI)] significantly enhance plasmid DNA condensation of low molecular weight (MW) PEIs. The star-block copolymers were prepared via a facile synthesis route using hexamethylene diisocyanate as linker between PEG and PEI blocks. NMR and FT-IR spectroscopy confirmed the structures of intermediately activated PEG and final products. Furthermore, the copolymers were characterized by size exclusion chromatography, static light scattering, and viscosimetry. Their molecular weights (M(w) 19-26 kDa) were similar to high MW PEI (25 kDa). Thermoanalytical investigations (thermogravimetric analysis, differential scanning calorimetry) were also performed and verified successful copolymer synthesis. DNA condensation with the low MW PEIs (800 and 2000 Da) and their 4- and 8-star-block copolymers was studied using atomic force microscopy, dynamic light scattering, zeta-potential measurements, and ethidium bromide (EtBr) exclusion assay. It was found that low MW PEIs formed huge aggregates (500 nm to 2 microm) in which DNA is only loosely condensed. By contrast, the star-block copolymers yielded small (80-110 nm), spherical and compact complexes that were stable against aggregation even at high ionic strength and charge neutrality. Furthermore, as revealed in the EtBr exclusion assay these star-block copolymers exhibited a DNA condensation potential as high as high MW PEI. Since these star-(PEG-block-PEI) copolymers are composed of relatively nontoxic low MW PEI and biocompatible PEG, their potential as gene delivery agents merits further investigations.     

Phosphorus NMR spectra of natural DNA fragments in the course of the B-to-A conformational transition

Changes in the ³¹P-nmr spectra of sonicated natural DNA fragments were investigated in ethanol solutions where the fragments underwent, as checked by CD, the B-to-A conformational transition. The study produced the following conclusions: (1) The high DNA concentrations used for the ³¹P-nmr measurements promote the transition compared to dilute solutions that are commonly used for CD measurements. (2) The B-to-A transition was reflected in a cooperative downfield shift of the DNA ³¹P-nmr resonance, consistent with unwinding of the double helix. (3) Prior to the transition, the changes in chemical shift of double-and single-stranded DNAs were almost identical. It thus appears that the effect of ethanol on the geometry and hydration of phosphodiester linkages does not depend heavily on DNA base–base interactions. (4) The A-form resonances were 30–40% narrower than the B-form resonances, which is attributed to marked sequence-dependent variations in the latter conformation and to their reduction in the former. (5) The B-form DNA aggregated in the concentrated ³¹P-nmr samples in the presence of ethanol, judged from a milky opalescence of the solution and a substantial broadening of its ³¹P-nmr resonance. The broadening abruptly disappeared as soon as DNA adopted the A-form so that DNA, in dependence on the secondary structure, showed different tendencies to condense in the presence of ethanol. The condensation increased cooperativity of the B-to-A interconversion.     

Structural alteration from non-B to B-form could reflect DNase I hypersensitivity

Preferential cleavage of active genes by DNase I has been correlated with a structurally altered conformation of DNA at the hypersensitive site in chromatin. To have a better understanding of the structural requirements for gene activation as probed by DNase I action, digestability by DNase I of synthetic polynucleotides having the ability to adopt B and non-B conformation (like Z-form) was studied which indicated a marked higher digestability of the B-form of DNA. Left handed Z form present within a natural sequence in supercoiled plasmid also showed marked resistance towards DNase I digestion. We show that alternating purine-pyrimidine sequences adopting Z-conformation exhibit DNAse I foot printing even in a protein free system. The logical deductions from the results indicate that 1) altered structure like Z-DNA is not a favourable substrate for DNase I, 2) both the ends of the alternating purine-pyrimidine insert showed hypersensitivity, 3) B-form with a minor groove of 12-13 A is a more favourable substrate for DNase I than an altered structure, 4) any structure of DNA deviating largely from B form with a capacity to flip over to the B-form are potential targets for the DNase I enzymic probes in naked DNA.     

Conformational analysis of d(C3G3), a B-family duplex in solution

NMR and circular dichroism studies of the duplex formed by the self-complementary DNA hexanucleotide d(C3G3) indicate that it is a B-type structure but differs from standard B-form. An analysis of NMR coupling constants within the deoxyribose moieties yields a 70% or greater contribution from pseudorotation phase angles corresponding to the C3'-exo conformation, a conformation similar to the C2'-endo conformation associated with B-form DNA. Intranucleotide interproton distances are consistent with a B-form structure, but some internucleotide distances are intermediate between A- and B-form structures. Circular dichroism spectra have B-form characteristics but also include an unusual negative band at 282 nm. The solution spectroscopic results are in contrast with X-ray crystallographic studies which find A-form structures for similar sequences.