Base protonation facilitates B-Z interconversions of poly(dG-dC) X poly(dG-dC)

Comparative studies on the salt titration and the related kinetics for poly(dG-dC) X poly(dG-dC) in pH 7.0 and 3.8 solutions clearly suggest that base protonation facilitates the kinetics of B-Z interconversion although the midpoint for such a transition in acidic solution (2.0-2.1 M NaCl) is only slightly lower than that of neutral pH. The rates for the salt-induced B to Z and the reverse actinomycin D induced Z to B transitions in pH 3.8 solutions are at least 1 order of magnitude faster than the corresponding pH 7.0 counterparts. The lowering of the B-Z transition barrier is most likely the consequence of duplex destabilization due to protonation as indicated by a striking decrease (approximately 40 degrees C) in melting temperature upon H+ binding in low salt. The thermal denaturation curve for poly(dG-dC) X poly(dG-dC) in a pH 3.8, 2.6 M NaCl solution indicates an extremely cooperative melting at 60.5 degrees C for protonated Z DNA, which is immediately followed by aggregate formation and subsequent hydrolysis to nucleotides at higher temperatures. The corresponding protonated B-form poly(dG-dC) X poly(dG-dC) in 1 M NaCl solution exhibits a melting temperature about 15 degrees C higher, suggesting further duplex destabilization upon Z formation.     

Specific and nonspecific interactions of integration host factor with oligo DNAs as revealed by circular dichroism spectroscopy and filter binding assay.

Binding specificity of integration host factor (IHF) to oligo DNAs has been studied by circular dichroism (CD) spectroscopy and filter binding experiment. CD difference spectra of IHF-DNA complexes demonstrated that a conformational change in DNA was induced by binding of IHF when DNA had a consensus sequence for the binding sites of IHF, but that such conformational change was not observed for consensus DNA 20 mer as well as nonconsensus DNA 45 mer. Dissociation constants for IHF-DNA complexes determined by filter binding assay showed that IHF has indeed stronger affinity to DNA with the consensus binding site than to nonconsensus DNA, but the difference in its affinity between consensus and nonconsensus DNAs was rather small, 3.4-fold. It was, therefore, concluded that the flanking regions of the consensus sequence are important for the specific binding of IHF and that its binding specificity is well characterized by the induced conformational change in DNA rather than by dissociation constants for IHF-DNA complexes.     

Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA

We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites.     

1H-NMR Investigation of the Interaction of the Amino Terminal Domain of the LexA Repressor with a Synthetic Half-Operator

Abstract

A synthetic half-operator DNA-duplex, d(GCTACTGTATGT), containing a portion of the proposed recognition sequence (CTGT) of serveral “SOS” genes, has been synthesized. The dodecamer has been characterized through ¹H-NMR spectroscopy. Complete assignment of exchangeable hydrogen bonded imino protons has been acheived by applying 1D NOE techniques and an analysis of the temperature dependence of the chemical shifts. In order to determine the specific role of the CTGT consensus sequence in the overall recognition process, the oligonucleotide duplex has been titrated with the amino terminal DNA binding domain of the LexA repressor. The observation of substantial changes of ¹H-NMR chemical shifts in the imino proton region upon interaction with the protein strongly suggests that the protein binds specifically to the operator DNA. The largest deviations of ¹H-NMR chemical shifts upon protein binding have been observed for protons assigned to the CTGT segment, thus strongly suggesting a direct involvement of this sequence in the binding process. At high potassium chloride concentrations the ¹H-NMR chemical shift deviations are reverted which is consistent with the known drop in the affinity constant of LexA for operator DNA at high salt concentrations.     

Absorption and fluorescence studies of the binding of the recA gene product from E. coli to single-stranded and double-stranded DNA. Ionic strength dependence

The binding of the recA gene product from E. coli to double-stranded and single-stranded nucleic acids has been investigated by following the change in melting temperature of duplex DNA and the fluorescence of single-stranded DNA or poly(dA) modified by reaction with chloroacetaldehyde. At low ionic strength, in the absence of Mg2+ ions, RecA protein binds preferentially to duplex DNA or poly(dA-dT). This leads to an increase of the DNA melting temperature. Stabilization of duplex DNA decreases when ionic strength or pH increases. In the presence of Mg2+ ions, preferential binding to single-stranded polynucleotides is observed. Precipitation occurs when duplex DNA begins to melt in the presence of RecA protein. From competition experiments, different single-stranded and double-stranded polydeoxynucleotides can be ranked according to their ability to bind RecA protein. Structural changes induced in nucleic acids upon RecA binding are discussed together with conformational changes induced in RecA protein upon magnesium binding.     

Binding of pyrene to DNA, base sequence specificity and its implication.

Solubilization as well as spectral studies of pyrene in natural DNA and synthetic deoxypolynucleotide solutions at neutral pH reveal at least two binding modes. Sites I are predominant in native DNA and in poly(dA-dT): poly(dA-dT) whereas sites II are found with denatured DNA and other polynucleotides such as poly(dA):poly(dT) and three different types of guanine containing copolymers which solubilize pyrene to a lesser extent. Spectral comparison with the covalent adducts of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydro-benzo(a)pyrene (anti-BPDE) and the physical complexes of its tetraols lead to the suggestion of a base sequence specific binding model for this carcinogenic metabolite to account for the puzzling fact that although its physical binding is predominantly intercalative, the covalent adducts appear not to be intercalated. It is speculated that in neutral solutions, intercalation may have little, if any, to do with the chemical lesion of this metabolite to the guanine base of the DNA and may, on the contrary, provide an efficient pathway for detoxification.     

Pyrene is highly emissive when attached to the RNA duplex but not to the DNA duplex: the structural basis of this difference

Through binding and fluorescence studies of oligonucleotides covalently attached to a pyrene group via one carbon linker at the sugar residue, we previously found that pyrene-modified RNA oligonucleotides do not emit well in the single-stranded form, yet the attached pyrene emits with a significantly high quantum yield upon binding to a complementary RNA strand. In sharp contrast, similarly modified pyrene–DNA probes exhibit very weak fluorescence both in the double-stranded and single-stranded forms. The pyrene-modified RNA oligonucleotides therefore provide a useful tool for monitoring RNA hybridization. The purpose of this paper is to present the structural basis for the different fluorescence properties of pyrene-modified RNA/RNA and pyrene-modified DNA/DNA duplexes. The results of absorption, fluorescence anisotropy and circular dichroism studies all consistently indicated that the pyrene attached to the RNA duplex is located outside of the duplex, whereas the pyrene incorporated into the DNA duplex intercalates into the double helix. ¹H NMR measurements unambiguously confirmed that the pyrene attached to the DNA duplex indeed intercalates between the base pairs of the duplex. Molecular dynamics simulations support these differences in the local structural elements around the pyrene between the pyrene–RNA/RNA and the pyrene–DNA/DNA duplexes.     

A simple method for studying the solubilization of polycyclic aromatic hydrocarbons in DNA solutions

A simple method for detecting the amount of a polycyclic aromatic hydrocarbon (PAH) solubilized in DNA solutions is described. This method takes advantage of the fact that sodium dodecyl sulfate (SDS) induces dissociation of PAH from DNA, and the greatly enhanced solubility of PAH in SDS solution makes possible the direct preparation of a standard solution for the extinction coefficient determination. The ability to make direct comparison between the spectroscopic characteristics before and after dissociation is accomplished by utilizing a tandem cuvette. The usefulness of this technique is then demonstrated by applying it to solubilization studies of pyrene in native and denatured DNA solutions of varying sodium chloride concentrations as well as in deoxymononucleotide solutions. Binding constants are estimated and the solubility data for pyrene in the DNA solutions are interpreted in terms of the binding abilities of intercalative versus external sites and the effect of salt. The mononucleotide results suggest a binding preference of pyrene to purine bases. It was observed that the binding constant of mononucleotide to pyrene is an order of magnitude lower than that of single-stranded DNA, which in turn is an order of magnitude smaller than that of duplex DNA.     

Conformations of DNA strands containing GAGT, GACA, or GAGC tetranucleotide repeats

The (GA)(n) microsatellite has been known from previous studies to adopt unusual, ordered, cooperatively melting secondary structures in neutral aqueous solutions containing physiological concentrations of salts, at acid pH values or in aqueous ethanol solutions. To find more about the primary structure specificity of these structures, we performed parallel comparative studies of related tetranucleotide repeats (GAGC)(5), (GAGT)(5), and (GACA)(5). The general conclusion following from these comparative studies is that the primary structure specificity is fairly high, indicating that not only guanines but also adenines play a significant role in the stabilization of these unusual structures. (GAGC)(5) is a hairpin or a duplex depending on DNA concentration. Neither acid pH nor ionic strength or the presence of ethanol changed the secondary structure of (GAGC)(5) in a significant way. (GACA)(5) forms a weakly stable hairpin in neutral aqueous solutions but forms a duplex at acid pH where cytosine is protonated. (GAGT)(5) behaves most similar to (GAGA)(5). Salt induces its hairpin to duplex transition at neutral pH and an isomerization into another, probably parallel stranded, duplex takes place at acid pH. (GAGT)(5) is the only of the three present 20-mers that responds to ethanol like (GAGA)(5).     

Novel sequence-responding fluorescent oligoDNA probe bearing a silylated pyrene molecule

A novel fluorescent phosphoramidite derivative of dimethylsilylated pyrene was prepared and incorporated into oligoDNA. The fluorescent oligoDNA exhibited marked fluorescent signal upon binding to the fully matched complementary DNA strand, however, the signal was strongly quenched in the single-stranded form as well as in the duplex having mismatched base pair at the terminus of the duplex-forming region.     

N-phenyl-carbazole-based two-photon fluorescent probes: strong sequence dependence of the duplex vs quadruplex selectivity

Herein we report on the synthesis and DNA recognition properties of a series of three N-phenyl carbazole-based light-up probes initially designed for two-photon absorption. The vinylic derivatives (Cbz-2Py, Cbz-3Py) display strong fluorescence enhancement when bound to various duplex- and quadruplex-forming oligonucleotides whereas the oxazole derivative is not fluorescent in DNA. Determination of affinity constants by fluorimetric titrations evidenced that Cbz-2Py has a clear preference for AT-rich duplex structures. Circular Dichroism (CD) measurements confirmed the sequence-dependent binding of this compound and suggest insertion in the minor groove as shown by a strong induced CD (ICD) signal and further supported by molecular modeling. Altogether the data indicate that duplex vs quadruplex selectivity of the dyes is strongly dependent on the sequence of the duplex. Finally, the dyes exhibit high two-photon absorption cross-sections (up to 540 GM in glycerol) and allow a fine and bright staining of nuclear DNA with low background fluorescence as shown by one and two-photon confocal microscopy imaging of fixed cells.     

Chain length-dependent association of distamycin-type oligopeptides with A·T and G·C pairs in polydeoxynucleotide duplexes

Different binding affinities of various distamycin analogs including the deformylated derivative with poly(dA-dC)·poly(dG-dT) were investigated using CD measurements. The inhibitory effect of distamycins on the DNAase I cleavage activity of DNA duplexes strongly supports the binding data. The base specificity of the ligand interaction with duplex DNA depends on the chain length of distamycin analogs. Netropsin, distamycin-2 and the deformylated distamycin-3 show no binding to dG·dC containing sequences at moderate ionic strength and are classified as highly dA·dT specific. In contrast distamycin having three, four or five methylpyrrolecarboxamide groups also forms more or less stable complexes with dG·dC-containing duplexes. These ligands possess a lower basepair specificity. The correlation between binding behavior and oligopeptide structure shows that presence of the number of hydrogen acceptor and donor sites determines the basepair and sequence specificity. The additional interaction with dG·dC pairs becomes essential when the number of hydrogen acceptor sites exceeds n = 3.     

Structural changes induced by binding of the high-mobility group I protein to a mouse satellite DNA sequence

Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA complexes formed, as determined by Job plots derived from experiments using pyrene-labeled duplexes. Circular dichroism and melting temperature experiments extended in the far ultraviolet range show that while native HMG-I is mainly random coiled in solution, it adopts a beta-turn conformation upon forming a 1:1 complex in which the protein first binds to one of two dA.dT stretches present in the duplex. HMG-I structure in the 1:1 complex is dependent on the sequence of its DNA target. A 3:1 HMG-I/DNA complex can also form and is characterized by a small increase in the DNA natural bend and/or compaction coupled to a change in the protein conformation, as determined from fluorescence resonance energy transfer (FRET) experiments. In addition, a peptide corresponding to an extended DNA-binding domain of HMG-I induces an ordered condensation of DNA duplexes. Based on the constraints derived from pyrene excimer measurements, we present a model of these nucleated structures. Our results illustrate an extreme case of protein structure induced by DNA conformation that may bear on the evolutionary conservation of the DNA-binding motifs of HMG-I. We discuss the functional relevance of the structural flexibility of HMG-I associated with the nature of its DNA targets and the implications of the binding stoichiometry for several aspects of chromatin structure and gene regulation.     

The Absence of Non-Local Conformational Changes in OR3 Operator DNA on Complexing with the Cro Repressor

Abstract

The Interaction of the cro protein of λ phage with a synthetic OR3 operator having 17 base pairs in length and with its 9 bp fragment has been studied using the circular dichroism (CD) method. In both cases, a considerable change in the CD of the samples was found in the region 260-300 nm upon the addition of the cro protein. The stoichiometry obtained by the CD titration was identical for OR3 and its 9 bp fragment: one duplex per dimeric cro.

NaCl addition makes the complexes dissociate so that the 9 bp fragment becomes free at [NaCl]>0.2 M while the whole OR3 becomes free at [NaCl]>0.5 M.

The CD spectra of both the free duplexes show a typical B-form conservative pattern with a positive CD band (270 nm) and a negative one (250 nm). The specific complexing of both the duplexes results in a substantial CD depression in the positive band. The most pronounced effect occurs at 280 nm. This spectral change is quite distinct from those in the B to A transition and in the non-cooperative winding of the DNA within the B-family of forms.

The interaction of the cro protein with the non-operator DNAs, calf thymus DNA and a synthetic 10 bp duplex, reveals no visible CD changes at all.

An inference is drawn that the CD change in the specific complexes is mainly due to the induced CD in tyr-26 upon its interaction with a specific base pair in the operator or its fragment, the operator DNA conformation being conserved in a B-like form as a whole. However, some local distortions such as kinks cannot be ruled out on the basis of the CD data.     

Groove Width and Depth of B-DNA Structures Depend on Local Variation in Slide

Abstract

The groove widths of DNA helix, especially minor groove width, are generally believed to be important for recognition of DNA by various types of ligands. It has been postulated earlier that large negative propeller twist, in the AT rich regions compresses the minor groove of duplex DNA A systematic study has now been carried out by generating models with different values of local doublet and intra-basepair parameters and calculating their minor groove widths. It is found that several local doublet parameters affect the minor groove width but it depends most strongly on the local step parameters roll and slide when each parameter is considered individually. However, a detailed analysis of the various local parameters within the B-DNA family of crystal structures indicates that propeller twist and slide are most strongly correlated with the observed values of minor groove width. The groove depth is also strongly correlated with slide. Thus the local base sequence dependent variations in slide can modify both the groove width and depth and consequently determine the ligand binding properties of DNA.     

Binding of Nonintercalative Antitumor Drugs to DNA-Polymers: Structural Effects of Bisquaternary Ammonium Heterocycles

Abstract

The binding of the antitumor agents SN-16814 nd SN-13232 to various DNA's in solution was monitored by CD and UV absorption measurements. In addition comparative studies with dA · dT containing duplex DNA of the related ligands SN-6136 and SN-6324 were included with respect to effects of structural variations. In general all four ligands show a dA · dT preference in their binding affinity to DNA.

Differences were observed for the reaction of SN-16814 which contains bicyclic ring system: it has a lower base pair selectivity, shows some affinity to poly(dG-dC) · poly(dG-dC), poly(rA) · poly(rU) and poly(rU). The binding mechanism of SN-16814 is associated with a significant time dependent binding effect in CD spectra and UV absorption in case of reaction with poly(dA) · poly(dT) and poly(dI) · poly(dC) indicating a slow kinetics.

The preferred binding to dA · dT base pairs in DNA decreases in the order from SN-61367 > SN-13232 > SN-6324, SN-16814 as judged from CD titration studies, salt dissociation and melting temperature data. Competitive binding experiments with netropsin (Nt) or distamycin-5 revealed that SN-16814 and SN-13232 are displaced from poly(dA-dT) · poly(dA-dT) suggesting that both ligands are less strongly bound than Nt and Dst-5 within the minor groove of B-DNA. These studies are consistent with results of the DNAase I cleavage of poly(dA-dT) · poly(dA-dT) which show the same relative order of inhibition of the cleavage reaction due to ligand binding. The results suggest that the variability of the DNAbinding and dA · dT sequence specificity may reside in the adaptability of benzamide-type ligands in the helical groove which is influenced by distinct structural modifications of the ligand conformation.     

Chain length-dependent association of distamycin-type oligopeptides with A X T and G X C pairs in polydeoxynucleotide duplexes

Different binding affinities of various distamycin analogs including the deformylated derivative with poly(dA-dC) X poly(dG-dT) were investigated using CD measurements. The inhibitory effect of distamycins on the DNAase I cleavage activity of DNA duplexes strongly supports the binding data. The base specificity of the ligand interaction with duplex DNA depends on the chain length of distamycin analogs. Netropsin, distamycin-2 and the deformylated distamycin-3 show no binding to dG X dC containing sequences at moderate ionic strength and are classified as highly dA X dT specific. In contrast distamycin having three, four or five methylpyrrolecarboxamide groups also forms more or less stable complexes with dG X dC-containing duplexes. These ligands possess a lower basepair specificity. The correlation between binding behavior and oligopeptide structure shows that presence of the number of hydrogen acceptor and donor sites determines the basepair and sequence specificity. The additional interaction with dG X dC pairs becomes essential when the number of hydrogen acceptor sites exceeds n = 3.     

Interaction of the antitumour alkaloid coralyne with duplex deoxyribonucleic acid structures: spectroscopic and viscometric studies.

The interaction of coralyne, an antitumour alkaloid with natural and synthetic duplex DNAs was investigated under conditions where the drug existed fully as a true monomer for the first time using spectrophotometric, spectrofluorimetric, circular dichroic and viscometric techniques. The absorption spectrum of coralyne monomer showed hypochromic and bathochromic effects on binding to duplex DNAs. This effect was used to determine the binding parameters of coralyne. The binding constants for four natural DNAs and four synthetic polynucleotides obtained from spectrophotometric titration, according to an excluded site model, using McGhee-von Hippel analysis, were all in the range of (0.38-9.8) x 10(5) M-1, and showed a relatively high specificity for the GC rich ML DNA and the alternating GC polynucleotide. The binding of coralyne decreased with increasing ionic strength, indicating that the binding affinity has a strong electrostatic component. Coralyne stabilized all the DNAs against thermal strand separation. The intense steady state fluorescence of coralyne was effectively quenched on binding to DNAs and the quantitative data on the Stern-Volmer quenching constant obtained was sequence dependent, being maximum with the GC rich DNA and alternating GC polymer. Circular dichriosm studies further evidenced for a strong perturbation of the B-conformation of DNAs consequent to coralyne binding with the concomitant development of extrinsic circular dichroic bands for the bound drug molecules suggesting their strong intercalated geometry in duplex DNAs. Further tests of intercalation using viscosity measurements on linear and covalently closed plasmid DNA conclusively proved the strong intercalation of coralyne in duplex DNA. Binding of the closely related natural alkaloid, berberine under these conditions showed considerably lower affinity to duplex DNAs in all experiments. Taken together, these results suggest that coralyne binds strongly to duplex DNAs by a mechanism of intercalation with specificity towards alternating GC duplex structure.     

Binding of Bis-linked Netropsin Derivatives in the Parallel-Stranded Hairpin Form to DNA

Abstract

Cis-diammine Pt(II)- bridged bis-netropsin and oligomethylene-bridged bis-netropsin in which two monomers are linked in a tail-to-tail manner bind to the DNA oligomer with the sequence 5′-CCTATATCC-3′ in a parallel-stranded hairpin form with a stoichiometry 1:1. The difference circular dichroism (CD) spectra characteristic of binding of these ligands in the hairpin form are similar. They differ from CD patterns obtained for binding to the same duplex of another bis-netropsin in which two netropsin moieties were linked in a head-to-tail manner. This reflects the fact that tail-to-tail and head-to-tail bis-netropsins use parallel and antiparallel side-by-side motifs, respectively, for binding to DNA in the hairpin forms. The binding affinity of cis -diammine Pt(II)- bridged bis-netropsin in the hairpin form to DNA oligomers with nucleotide sequences 5′-CCTATATCC-3′ (I), 5′-CCTTAATCC-3′ (II), 5′-CCTTATTCC-3′ (III), 5′-CCTTTTTCC-3′ (IV) and 5′-CCAATTTCC-3′ (V) decreases in the order I = II > III > IV> V. The binding of oligomethylene-bridged bis-netropsin in the hairpin form follows a similar hierarchy. An opposite order of sequence preferences is observed for partially bonded monodentate binding mode of the synthetic ligand.