Temperature-dependent ultraviolet resonance Raman spectroscopy of the premelting state of dA.dT DNA.

Poly(dA).poly(dT) and DNA duplex with four or more adenine bases in a row exhibits a broad, solid-state structural premelting transition at about 35 degrees C. The low-temperature structure is correlated with the phenomena of "bent DNA." We have conducted temperature-dependent ultraviolet resonance Raman measurements of the structural transition using poly(dA).poly(dT) at physiological salt conditions, and are able to identify, between the high and low temperature limits, changes in the vibrational frequencies associated with the C4 carbonyl stretching mode in the thymine ring and the N6 scissors mode of the amine in the adenine ring of poly(dA).poly(dT). This work supports the model that the oligo-dA tracts' solid-state structural premelting transition is due to a set of cross-stand bifurcated hydrogen bonds between consecutive dA. dT pairs.     

Differences in melting behavior between homopolymers and copolymers of DNA: Role of nonbonded forces for GC and the role of the hydration spine and premelting transition for AT

Melting measurements of the mono-base-pair DNA polymers showed that the melting temperature T_m of the B-DNA homopolymer poly (dA ) · poly (dT) is higher than that of the copolymer poly [d(A-T)]. On the other hand, the T_mof the B-DNA homopolymer poly (dG) · poly (dC) is lower than that of the copolymer poly [d (G-C)]. From a structural point of view, the cross-strand base-stacking interaction in a DNA homopolymer is weaker than that in a DNA copolymer with the same base pair. One would then expect that all the DNA homopolymers are less stable than the copolymer with the same base pair. We find that the inversion of the melting order seen in the AT mono-base-pair DNA polymers is caused by the enhanced thermal stability of poly (dA) · poly (dT) from a well-defined spine of hydration attached to its minor groove. In this paper we employ the modified self-consistent phonon theory to calculate base-pair opening probabilities of four B-DNA polymers: poly(dA)-poly(dT), poly(dG) · poly(dC), poly[d(A-T)], and poly[d(G-C)] at temperatures from room temperature through the melting regions. Our calculations show that the spine of hydration can give the inverted melting order of the AT polymers as compared to the GC polymers in fair agreement with experimental measurements. Our calculated hydration spine disruption behavior in poly(dA) · poly(dT) at premelting temperatures is also in agreement with experimentally observed premelting transitions in poly (dA) · poly (dT). The work is in a sense a test of the validity of our models of nonbonded interactions and spine of hydration interactions. We find we have to develop the concept of a strained bond to fit observations in poly (dA) · poly(dT). The strained-bond concept also explains the otherwise anomalous stability of the hydration chain. © 1993 John Wiley & Sons, Inc.     

Determination of the kinetics of deuteration of DNA.RNA hybrids by ultraviolet spectroscopy

The kinetics of the hydrogen-deuterium exchange reactions of poly(dA).poly(rU) and poly(rA).poly(dT) has been examined, at pH 7.0 and at various temperatures in the 15-35 degrees C range, by stopped-flow ultraviolet spectrophotometry. For comparison, the deuteration kinetics of poly[d(A-T)].poly[d(A-T)] and poly(rA).poly(rU) has been reexamined. At 20 degrees C, the imino deuteration (NH----ND) rates of the two hybrid duplexes were found to be 1.5 and 1.8 s-1, respectively. These are nearly equal to the imino deuteration rates of poly[d(A-T)].poly[d(A-T)] (1.1 s-1) and poly(rA).poly(rU) (1.5 s-1) but appreciably higher than that of poly(dA).poly(dT) (0.35 s-1). It has been suggested that a DNA.RNA hybrid, an RNA duplex, and the AT-alternating DNA duplex have in general higher base-pair-opening reaction rates than the ordinary DNA duplex. The amino deuteration (NH2----ND2) rates, on the other hand, have been found to be 0.25, 0.28, and 0.33 s-1, respectively, for poly(dA).poly(rU), poly(rA).poly(dT), and poly[d(A-T)].poly[d(A-T)], at 20 degrees C. These are appreciably higher than that for poly(rA).poly(rU) (0.10 s-1). In general, the equilibrium constants (K) of the base-pair opening are considered to be greatest for the DNA.RNA hybrid duplex (0.05 at 20 degrees C), second greatest for the RNA duplex (0.02 at 20 degrees C), and smallest for the DNA duplex (0.005 at 20 degrees C), although the AT-alternating DNA duplex has an exceptionally great K (0.07 at 20 degrees C). From the temperature effect on the K value, the enthalpy of the base-pair opening was estimated to be 3.0 kcal/mol for the DNA.RNA hybrid duplex.     

UV resonance Raman and circular dichroism studies of a DNA duplex containing an A(3)T(3) tract: evidence for a premelting transition and three-centered H-bonds.

The presence of A(n) and A(n)T(n) tracts in double-helical sequences perturbs the structural properties of DNA molecules, resulting in the formation of an alternate conformation to standard B-DNA known as B'-DNA. Evidence for a transition occurring prior to duplex melting in molecules containing A(n) tracts was previously detected by circular dichroism (CD) and calorimetric studies. This premelting transition was attributed to a conformational change from B'- to B-DNA. Structural features of A(n) and A(n)T(n) tracts revealed by X-ray crystallography include a large degree of propeller twisting of adenine bases, narrowed minor grooves, and the formation of three-centered H-bonds between dA and dT bases. We report UV resonance Raman (UVRR) and CD spectroscopic studies of two related DNA dodecamer duplexes, d(CGCAAATTTGCG)(2) (A(3)T(3)) and d(CGCATATATGCG)(2) [(AT)(3)]. These studies address the presence of three-centered H-bonds in the B' conformation and gauge the impact of these putative H-bonds on the structural and thermodynamic properties of the A(3)T(3) duplex. UVRR and CD spectra reveal that the premelting transition is only observed for the A(3)T(3) duplex, is primarily localized to the dA and dT bases, and is associated with base stacking interactions. Spectroscopic changes associated with the premelting transition are not readily detectable for the sugar-phosphate backbone or the cytosine and guanosine bases. The temperature-dependent concerted frequency shifts of dA exocyclic NH(2) and dT C4=O vibrational modes suggest that the A(3)T(3) duplex forms three-centered hydrogen bonds at low temperatures, while the (AT)(3) duplex does not. The enthalpy of this H-bond, estimated from the thermally induced frequency shift of the dT C4=O vibrational mode, is approximately 1.9 kJ/mol or 0.46 kcal/mol.     

Temperature dependence of the Raman spectrum of DNA. II. Raman signatures of premelting and melting transitions of poly(dA).poly(dT) and comparison with poly(dA-dT).poly(dA-dT).

The temperature dependence of the Raman spectrum of poly(dA).poly(dT) (dA: deoxyadenosine; dT: thymidine), a model for DNA containing consecutive adenine.thymine (A.T) pairs, has been analyzed using a spectrometer of high spectral precision and sensitivity. Three temperature intervals are distinguished: (a) premelting (10 < t < 70 degrees C), in which the native double helix is structurally altered but not dissociated into single strands; (b) melting (70 < t < 80 degrees C), in which the duplex is dissociated into single strands; and (c) postmelting (80 < t degrees C), in which no significant structural change can be detected. The distinctive Raman difference signatures observed between 10 and 70 degrees C and between 70 and 80 degrees C are interpreted in terms of the structural changes specific to premelting and melting transitions, respectively. Premelting alters the low-temperature conformation of the deoxyribose-phosphate backbone and eliminates base hydrogen bonding that is distinct from canonical Watson-Crick hydrogen bonding; these premelting perturbations occur without disruption of base stacking. Conversely, melting eliminates canonical Watson-Crick pairing and base stacking. The results are compared with those reported previously on poly(dA-dT).poly(dA-dT), the DNA structure consisting of alternating A.T and T.A pairs (L. Movileanu, J. M. Benevides, and G. J. Thomas, Jr. Journal of Raman Spectroscopy, 1999, Vol. 30, pp. 637-649). Poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) exhibit strikingly dissimilar temperature-dependent Raman profiles prior to the onset of melting. However, the two duplexes exhibit very similar melting transitions, including the same Raman indicators of ruptured Watson-Crick pairing, base unstacking and collapse of backbone order. A detailed analysis of the data provides a comprehensive Raman assignment scheme for adenosine and thymidine residues of B-DNA, delineates Raman markers diagnostic of consecutive A.T and alternating A.T/T.A tracts of DNA, and identifies the distinct Raman difference signatures for premelting and melting transitions in the two types of sequences.     

The thermodynamic contribution of the 5-methyl group of thymine in the two- and three-stranded complexes formed by poly(dU) and poly(dT) with poly(dA)

To assess the thermodynamic contribution of the 5-methyl group of thymine, we have studied the two-stranded helical complexes poly(dA).poly(dU) and poly(dA).poly(dT) and the three-stranded complexes--poly(dA).2poly(dU), poly(dA).poly(dT).poly(dU) and poly(dA).2poly(dT)--by differential scanning calorimetry, and uv optical melting experiments. The thermodynamic quantities associated with the 3 --> 2, 2 --> 1, and 3 --> 1 melting transitions are found to vary with salt concentration and temperature in a more complex manner than commonly believed. The transition temperatures, T(m), are generally not linear in the logarithm of concentration or activity of NaCl. The change in enthalpy and in entropy upon melting varies with salt concentration and temperature, and a change in heat capacity accompanies each transition. The poly(dA).2poly(dU) triple helix is markedly different from poly(dA).2poly(dT) in both its CD spectrum and thermodynamic behavior, while the poly(dA).poly(dT).poly(dU) triple helix resembles poly(dA).2poly(dT) in these properties. In comparing poly(dA).2poly(dT) with either the poly(dA).poly(dT).poly(dU) or the poly(dA).2poly(dU) triplexes, the substitution of thymine for uracil in the third strand results in an enhancement of stability against the 3 --> 2 dissociation of deltadeltaG degrees = -135 +/- 85 cal (mol A)(-1) at 37 degrees C. This represents a doubling of the absolute stability toward dissociation compared to the triplexes with poly(dU) as the third strand. The poly (dA).poly (dT) duplex is more stable than poly(dA).poly(dU) by deltadeltaG degrees = -350 +/- 60 cal (mol base pair)(-1) at 37 degrees C. Poly(dA).poly(dT) has 50% greater stability than poly(dA).poly(dU) as a result of the dT for dU substitution in the duplex.     

Temperature dependence of the volumetric parameters of drug binding to poly[d(A-T)].Poly[d(A-T)] and Poly(dA).Poly(dT)

We report the temperature and salt dependence of the volume change (DeltaVb) associated with the binding of ethidium bromide and netropsin with poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)]. The DeltaV(b) of binding of ethidium with poly(dA).poly(dT) was much more negative at temperatures approximately 70 degrees C than at 25 degrees C, whereas the difference is much smaller in the case of binding with poly[d(A-T)].poly[d(A-T)]. We also determined the volume change of DNA-drug interaction by comparing the volume change of melting of DNA duplex and DNA-drug complex. The DNA-drug complexes display helix-coil transition temperatures (Tm several degrees above those of the unbound polymers, e.g., the Tm of the netropsin complex with poly(dA)poly(dT) is 106 degrees C. The results for the binding of ethidium with poly[d(A-T)].poly[d(A-T)] were accurately described by scaled particle theory. However, this analysis did not yield results consistent with our data for ethidium binding with poly(dA).poly(dT). We hypothesize that heat-induced changes in conformation and hydration of this polymer are responsible for this behavior. The volumetric properties of poly(dA).poly(dT) become similar to those of poly[d(A-T)].poly[d(A-T)] at higher temperatures.     

Probing the hydration of the minor groove of A.T synthetic DNA polymers by volume and heat changes

The minor-groove ligand netropsin provides a sensitive probe of the hydration difference between poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)]. We have measured the volume change delta V accompanying binding of netropsin to these polymers, using an improved magnetic suspension densimeter. For poly(dA).poly(dT) we find delta V = +97 mL/mol of bound netropsin at pH 7.0 and 10 mM sodium phosphate buffer. For poly[d(AT)].poly[d(AT)] we find delta V = -16 mL/mol of bound netropsin. This striking differential effect suggests that the poly(dA).poly(dT) duplex compresses more water (or is more extensively hydrated). From our enthalpy and entropy results we estimate the approximately 10 water molecules, immobilized in the minor groove of this system, are displaced by each netropsin bound. The volume increase, however, is substantially larger than can be explained by a simple melting of these immobilized water molecules in the minor groove. A decompression of at least 40 water molecules must attend the complexation to the poly(dA).poly(dT) duplex. This suggests that the conformation change attending the binding of the drug to this polymer duplex causes a further dehydration, whereas no such change in dehydration and configuration for the heteropolymer system is indicated.     

Circular dichroism of adenine and thymine containing synthetic polynucleotides

Circular dichroism (CD) spectra of poly(dA), poly(dT), poly(dA)·poly(dT), and poly[d(A-T)]·poly[d(T-A)] have been measured as a function of temperature. From these data difference spectra have been calculated by subtracting the spectrum measured at low temperature from the spectra measured at higher temperatures. The CD difference spectra obtained upon melting of the two double-stranded polymers are very similar. From a comparison of these difference spectra with calculated ones it is shown that optical transitions near 272 nm (on A) and 288 nm (most probably on T) are present. The premelting changes of the CD spectrum of poly[d(A-t)]·poly[d(T-A)] are due to a change in conformation in which the secondary structure goes from a C- to B-type spectrum by increasing the A-type nature of the polymer. Such a change is not observed for poly(dA)·poly(dT). Instead, a transition between two different B-type geometries occurs.     

Preferential binding of quinolones to DNA with alternating G,C / A,T sequences: a spectroscopic study

The binding of quinolones, nalidixic acid (Nal), oxolinic acid (Oxo) with double stranded polynucleotides was undertaken by using UV-melting, UV-Vis absorption, fluorescence and CD spectroscopic techniques. The binding of Nal or Oxo to the polynucleotides under low-salt buffer conditions were determined for poly (dA).(dT), poly [d(A-T)], poly (dG).(dC), poly [d(G-C)] and E. coli DNA. The fluorescence data were analyzed using a previously established two step mechanism with two different DNA-Drug complexes [Rajeswari et al., Biochemistry 26, 6825-31 (1987)]. The first complex [DN](1) with a binding constant K(1), is formed where the interactions are 'nonspecific' and complex [DN](2) with a binding constant K(2), is formed where the interactions are "specific" which involve (additional) hydrophobic type of interactions like 'stacking' of the drug and the overall association constant is represented as K(=K(1)K(2)). The order of binding for Nal and Oxo is: poly [d(G-C)] > poly [d(A- T)] > E. coli > poly (dG).(dC) > poly (dA).(dT). Interaction of quinolones seems to be preferential in the alternating G, C or A, T stretches of DNA than those of non-alternating. Within any alternating or non-alternating in DNA sequences the G, C rich sequences have distinctly greater binding than A, T sequences. The overall association constant data (K) reveal higher binding of Oxo to DNA compared to Nal to any given polynucleotide investigated; which also explains the higher antibacterial potency of Oxo. Changes in the absorption difference spectra and in circular dichroic spectra also manifest these results. As the melting temperatures of the polynucleotides were only marginally raised in presence of the quinolone, we rule out the possibility of 'classical intercalation' of the drug. Amino group of guanine facilitates the binding of quinolones and therefore has the greater binding with the DNA. However, poly (dG).(dC) is known to exist in 'A' conformation which is not adopted by quinolones as in the case of poly (dA).(dT). Present results suggest that Nal or Oxo bind to DNA in a non-classical fashion which is partially stacking in nature.     

fd gene 5 protein binds to double-stranded polydeoxyribonucleotides poly(dA.dT) and poly[d(A-T).d(A-T)]

Circular dichroism (CD) data indicated that fd gene 5 protein (G5P) formed complexes with double-stranded poly(dA.dT) and poly[d(A-T).d(A-T)]. CD spectra of both polymers at wavelengths above 255 nm were altered upon protein binding. These spectral changes differed from those caused by strand separation. In addition, the tyrosyl 228-nm CD band of G5P decreased more than 65% upon binding of the protein to these double-stranded polymers. This reduction was significantly greater than that observed for binding to single-stranded poly(dA), poly(dT), and poly[d(A-T)] but was similar to that observed for binding of the protein to double-stranded RNA [Gray, C.W., Page, G.A., & Gray, D.M. (1984) J. Mol. Biol. 175, 553-559]. The decrease in melting temperature caused by the protein was twice as great for poly[d(A-T).d(A-T)] as for poly(dA.dT) in 5 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7. Upon heat denaturation of the poly(dA.dT)-G5P complex, CD spectra showed that single-stranded poly(dA) and poly(dT) formed complexes with the protein. The binding of gene 5 protein lowered the melting temperature of poly(dA.dT) by 10 degrees C in 5 mM Tris-HCl, pH 7, but after reducing the binding to the double-stranded form of the polymer by the addition of 0.1 M Na+, the melting temperature was lowered by approximately 30 degrees C. Since increasing the salt concentration decreases the affinity of G5P for the poly(dA) and poly(dT) single strands and increases the stability of the double-stranded polymer, the ability of the gene 5 protein to destabilize poly(dA.dT) appeared to be significantly affected by its binding to the double-stranded form of the polymer.     

Selectivity of spermine homologs on triplex DNA stabilization.

We synthesized seven homologs of spermine (H2N(CH2)3NH(CH2)nNH(CH2)3NH2, where n = 2-9; n = 4 for spermine) and studied their effects on melting temperature (Tm), conformation, and precipitation of poly(dA).2poly(dT). The triplex DNA melting temperature, Tm1 was 34.4 degrees C in the presence of 150 mM KCl. Addition of spermine homologs increased Tm1 in a concentration-dependent and structure-dependent manner, with 3-6-3 (n = 6) exerting optimal stabilization. The dTm1/dlog[polyamine] values were 9-24 for these compounds. The duplex melting temperature, Tm2 was insensitive to homolog concentration and structure, suggesting their ability to stabilize triplex DNA without altering the stability of the underlying duplex. Circular dichroism spectral studies revealed psi-DNA formation in a concentration-dependent and structure-dependent manner. Phase diagrams were constructed showing the critical ionic/polyamine concentrations stabilizing different structures. These compounds also exerted structural specificity effects on precipitating triplex DNA. These data provide new insights into the ionic/structural determinants affecting triplex DNA stability and indicate that 3-6-3 is an excellent ligand to stabilize poly(dA).2poly(dT) triplex DNA under physiologic ionic conditions for antigene therapeutics.     

Spectroscopic analysis of the interaction of Escherichia coli DNA-dependent RNA polymerase with T7 DNA and synthetic polynucleotides.

We have studied the circular dichroism and ultraviolet difference spectra of T7 bacteriophage DNA and various synthetic polynucleotides upon addition of Escherichia coli RNA polymerase. When RNA polymerase binds nonspecifically to T7 DNA, the CD spectrum shows a decrease in the maximum at 272 but no detectable changes in other regions of the spectrum. This CD change can be compared with those associated with known conformational changes in DNA. Nonspecific binding to RNA polymerase leads to an increase in the winding angle, theta, in T7 DNA. The CD and UV difference spectra for poly[d(A-T)] at 4 degrees C show similar effects. At 25 degrees C, binding of RNA polymerase to poly[d(A-T)] leads to hyperchromicity at 263 nm and to significant changes in CD. These effects are consistent with an opening of the double helix, i.e. melting of a short region of the DNA. The hyperchromicity observed at 263 nm for poly[d(A-T)] is used to determine the number of base pairs disrupted in the binding of RNA polymerase holoenzyme. The melting effect involves about 10 base pairs/RNA polymerase molecule. Changes in the CD of poly(dT) and poly(dA) on binding to RNA polymerase suggest an unstacking of the bases with a change in the backbone conformation. This is further confirmed by the UV difference spectra. We also show direct evidence for differences in the template binding site between holo- and core enzyme, presumably induced by the sigma subunit. By titration of the enzyme with poly(dT) the physical site size of RNA polymerase on single-stranded DNA is approximately equal to 30 bases for both holo- and core enzyme. Titration of poly[d(A-T)] with polymerase places the figure at approximately equal to 28 base pairs for double-stranded DNA.     

Counterion association with native and denatured nucleic acids: an experimental approach

The melting temperature of the poly(dA) . poly(dT) double helix is exquisitely sensitive to salt concentration, and the helix-to-coil transition is sharp. Modern calorimetric instrumentation allows this transition to be detected and characterized with high precision at extremely low duplex concentrations. We have taken advantage of these properties to show that this duplex can be used as a sensitive probe to detect and to characterize the influence of other solutes on solution properties. We demonstrate how the temperature associated with poly(dA) . poly(dT) melting can be used to define the change in bulk solution cation concentration imparted by the presence of other duplex and triplex solutes, in both their native and denatured states. We use this information to critically evaluate features of counterion condensation theory, as well as to illustrate "crosstalk" between different, non-contacting solute molecules. Specifically, we probe the melting of a synthetic homopolymer, poly(dA) . poly(dT), in the presence of excess genomic salmon sperm DNA, or in the presence of one of two synthetic RNA polymers (the poly(rA) . poly(rU) duplex or the poly(rU) . poly(rA) . poly(rU) triplex). We find that these additions cause a shift in the melting temperature of poly(dA) . poly(dT), which is proportional to the concentration of the added polymer and dependent on its conformational state (B versus A, native versus denatured, and triplex versus duplex). To a first approximation, the magnitude of the observed tm shift does not depend significantly on whether the added polymer is RNA or DNA, but it does depend on the number of strands making up the helix of the added polymer. We ascribe the observed changes in melting temperature of poly(dA) . poly(dT) to the increase in ionic strength of the bulk solution brought about by the presence of the added nucleic acid and its associated counterions. We refer to this communication between non-contacting biopolymers in solution as solvent-mediated crosstalk. By comparison with a known standard curve of tm versus log[Na+] for poly(dA) . poly(dT), we estimate the magnitude of the apparent change in ionic strength resulting from the presence of the bulk nucleic acid, and we compare these results with predictions from theory. We find that current theoretical considerations correctly predict the direction of the t(m) shift (the melting temperature increases), while overestimating its magnitude. Specifically, we observe an apparent increase in ionic strength equal to 5% of the concentration of the added duplex DNA or RNA (in mol phosphate), and an additional apparent increase of about 9.5 % of the nucleic acid concentration (mol phosphate) upon denaturation of the added DNA or RNA, yielding a total apparent increase of 14.5 %. For the poly(rU) . poly(rA) . poly(rU) triplex, the total apparent increase in ionic strength corresponds to about 13.6% of the amount of added triplex (moles phosphate). The effect we observe is due to coupled equilibria between the solute molecules mediated by modulations in cation concentration induced by the presence and/or the transition of one of the solute molecules. We note that our results are general, so one can use a different solute probe sensitive to proton binding to characterize subtle changes in solution pH induced by the presence of another solute in solution. We discuss some of the broader implications of these measurements/results in terms of nucleic acid melting in multicomponent systems, in terms of probing counterion environments, and in terms of potential regulatory mechanisms.     

Microcalorimetric investigation of DNA, poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] melting in the presence of water soluble (meso tetra (4 N oxyethylpyridyl) porphyrin) and its Zn complex

Thermodynamic parameters of melting process (DeltaHm, Tm, DeltaTm) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C)).poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased Tm of satellite fraction by 24 degrees C, but ZnTOEpyp(4), on the contrary, predominantly bound with AT-rich sites and increased DNA main stage Tm by 18 degrees C, and Tm of poly(dA)poly(dT) increased by 40 degrees C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes--strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode--strong binding--took place. The weak binding is characterized with shifting of Tm by some grades, and for the strong binding Tm shifts by approximately 30-40 degrees C. Invariability of DeltaHm of DNA and poly(dA)poly(dT), and sharp increase of Tm in the range of r from 0.08 to 0.25 for thymus DNA and 0.01-0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H2O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width DeltaTm caused by increase of added ZnTOEpyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which DeltaT approximately TGC-TAT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193-205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

Characterization of thermostable RecA protein and analysis of its interaction with single-stranded DNA.

Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 degrees C but not at 37 degrees C, although nucleoprotein complex was observed at both temperatures. ttRecA showed single-stranded DNA (ssDNA)-dependent ATPase activity, and its activity was maximal at 65 degrees C. The kinetic parameters, K(m) and kcat, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 mM and 0.60 s-1 at 65 degrees C, and 0.34 mM and 0.28 s-1 at 37 degrees C, respectively. Substrate cooperativity was observed at both temperatures, and the Hill coefficient was about 2. At 65 degrees C, all tested ssDNAs were able to stimulate the ATPase activity. The order of ATPase stimulation was: poly(dC) > poly(dT) > M13 ssDNA > poly(dA). Double-stranded DNAs (dsDNA), poly(dT).poly(dA) and M13 dsDNA, were unable to activate the enzyme at 65 degrees C. At 37 degrees C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability. At 25 degrees C, poly(dA) and M13 ssDNA gave circular dichroism (CD) peaks at around 192 nm, which reflect a particular structure of DNA. The conformation was changed by an upshift of temperature or binding to Escherichia coli RecA protein (ecRecA), but not to ttRecA. The dissociation constant between ecRecA and poly(dA) was estimated to be 44 microM at 25 degrees C by the change in the CD. These observations suggest that the capability to modify the conformation of ssDNA may be different between ttRecA and ecRecA. The specific structure of ssDNA was altered by heat or binding of ecRecA. After this alteration, ttRecA and ecRecA can express their activities at each physiological temperature.     

Sequence-dependant polymorphism of DNA duplex in solutions

Raman spectra of six synthetic polydeoxyribonucleotide duplexes with different base sequences have been examined in aqueous solutions with different salt or nucleotide concentrations. Detailed conformational differences have been indicated between B and Z forms of poly[d(G-C)] X poly[d(G-C)], between B forms of poly[d(G-C)] X poly[d(G-C)] and poly[d(G-m5C)] X poly[d(G-m5C)], between A and B forms of poly(dG) X poly(dC), between B and "CsF" forms of poly[d(A-T)] X poly[d(A-T)], between B forms of poly[d(A-U)] X poly[d(A-U)] and poly[d(A-T)] X poly[d(A-T)], and between low- and high-salt (CsF) forms of poly(dA) X poly(dT). The Raman spectrum of calf-thymus DNA in aqueous solution was also observed and was compared with the Raman spectra of its fibers in A, B, and C forms.     

Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

Interaction of the nonintercalative antitumour drugs SN-6999 and SN-18071 with DNA: influence of ligand structure on the binding specificity

Binding to DNA's of the non-intercalative ligands SN-6999 and SN-18071 has been studied by means of circular dichroism, UV absorption, thermal melting and for SN-6999 by viscosity measurements. Both antitumour drugs show a preference for dA.dT rich DNA's, but the base pair selectivity of SN-18071 is lower as indicated by some affinity to dG.dC containing duplex DNA. The dA.dT base pair specificity of SN-6999 is comparable to that of netropsin. It forms very stable complexes with dA.dT containing duplex DNA and competes with netropsin binding on DNA. The ligands SN-18071 and pentamidine are totally released from their complexes with poly(dA-dT).poly(dA-dT) by competitive netropsin binding. The results demonstrate that hydrogen bonding capacity of the ligand in addition to other factors strongly contribute to the base sequence specificity in the recognition process of the ligand with DNA. A binding model of SN-6999 with five dA.dT pairs in the minor groove of B-DNA is suggested.     

Binding of ligands to a one-dimensional heterogeneous lattice. III. Kinetic model and relaxation study of the interaction of tilorone with DNA and polynucleotides

A temperature-jump relaxation study of the interaction of tilorone with different polynucleotides and DNA has been performed. A single relaxation time, attributed to the intercalation step, has been observed in the case of poly[d(A-T)]·poly[d(A-T)], poly[d(A-C)]·poly[d(G-T)], poly[d(G-C)]·poly[d(G-C)], and poly(dG)·poly(dC). No intercalation into poly(dA)·poly(dT) occurs, and the interaction with poly(dG)·poly(dC) is different from what is observed with the other intercalating homopolymers. Refinement of the binding model is suggested from the analysis of the kinetic data. The relaxation curves obtained with DNA are well simulated based on a binding mechanism where DNA is considered a heterogeneous lattice and each type of site behaves as if it were located in the corresponding homopolymer. Poly(dA)·poly(dT) shows a unique behavior: studies of the effects of concentration and temperature indicate that tilorone acts as a probe of a process involving the polynucleotide alone. This process appears to be related to the dynamic structure of the nucleic acid and is detectable only when the bound dye is not intercalated.