Unwinding kinetics of cooperatively melting regions in DNA

Unwinding of a single cooperatively melting region of ColE1 DNA is investigated by a slow temperature jump in formamide–neutral buffer mixed solvent. The semilogarithmic plots of unwinding relaxation curves show a marked terminal linear region following the fast decay, which occurred within the temperature rise time (1 ～ 2 s). This longest relaxation is ascribed to the total unwinding of a single cooperatively melting region. The longest relaxation time, τ₁, is uniquely determined by the final equilibrium state and becomes shorter as the final temperature increases. Decrease in ionic strength makes τ₁ and its fractional amplitude increase, and the relaxation almost approaches single-exponential decay. The facts that (1) τ₁ of a single cooperatively melting region whose unwinding suffers larger frictional resistance does not always unwind more slowly, as was shown by the observations of τ₁'s of almost the same cooperatively melting region located at different positions on two linearized ColEl DNAs and of τ₁'s of two cooperatively melting regions on the same linearized ColEl DNA; (2) τ₁ has strong dependence on the equilibrium state after a temperature jump; and (3) the observed τ₁ is much longer than the expected time of the frictional barrier all demonstrate that the τ₁ is limited by chemical but not hydrodynamic processes. The detailed unwinding process of a single cooperatively melting region, elucidated by evidence of a negative apparent activation energy of the rewinding process and by extensive computer simulation of the equilibrium melting process, suggests that the local heterogeneity of G+C content in a cooperatively melting region, as well as its averaged G+C content, strongly affects its unwinding rate. The present study of a single cooperatively melting region is found to be useful to improve our understanding of the detailed mechanism of complex unwinding of large natural DNAs, in which many cooperatively melting regions unwind.     

The stability of DNA intrastrand cross-links of antitumor transplatin derivative containing non-bulky methylamine ligands

Oligonucleotides modified by clinically ineffective trans-diamminedichloridoplatinum(II) (transplatin) have been shown to be effective modulators of gene expression. This is so because in some nucleotide sequences the 1,3-GNG intrastrand adducts formed by transplatin in double-helical DNA readily rearrange into interstrand cross-links so that they can cross-link the oligonucleotides to their targets. On the other hand, in a number of other sequences these intrastrand adducts are relatively stable, which represents the major difficulty in the clinical use of the antisense transplatin-modified oligonucleotides. Therefore, we examined in this study, the stability of 1,3-GNG intrastrand adducts in double-helical DNA formed by a new antitumor derivative of transplatin, trans-[Pt(CH₃NH₂)₂Cl₂], in the sequence contexts in which transplatin formed relatively stable intrastrand cross-links which did not readily rearranged into interstrand cross-links. We have found that 1,3-GNG intrastrand adducts in double-helical DNA formed by trans-[Pt(CH₃NH₂)₂Cl₂] even in such sequences readily rearrange into interstrand cross-links. This work also suggests that an enhanced frequency of intrastrand cross-links yielded by trans-[Pt(CH₃NH₂)₂Cl₂] is a consequence of the fact that these DNA lesions considerably distort double-helical DNA in far more sequence contexts than parent transplatin. Our results suggest that trans-[Pt(CH₃NH₂)₂Cl₂]-modified oligonucleotides represent promising candidates for new agents in antisense or antigene approach.     

A critical evaluation of models for complex molecular dynamics: application of NMR studies of double- and single-stranded DNA

The nature of internal and overall motions in native (double-stranded) and denatured (single-stranded) DNA fragments 120–160 base pairs (bp) long is examined by molecular-dynamics modeling using ¹³C-nmr spin-relaxation data obtained over the frequency range of 37–125 MHz. The broad range of ¹³C frequencies is required to differentiate among various models. Relatively narrow linewidths, large nuclear Overhauser enhancements (NOEs), and short T₁ values all vary significantly with frequency and indicate the presence of rapid, restricted internal motions on the nanosecond time scale. For double-stranded DNA monomer fragments (147 bp, 24 Å diam at 32°C), the overall motion is that of an axially symmetric cylinder (τ_x = ～10^?6 s;τ_Z = ～1.8 × 10^?8s), which is in good agreement with values calculated from hydrodynamic theory (τ_x = ～1.8 × 10^?6 s; τ_Z = ～2.7 × 10^?8 s). The DNA internal motion can be modeled as restricted amplitude internal diffusion of individual C? H vectors of deoxyribose methine carbons C1′, C3′, and C4′, either with conic boundary conditions (τ_w = ～4 × 10^?9 s, θ_cone = ～21°) or as a bistable jump (τ_A = τ_B = ～2 × 10^?9 s, θ = ～15°). We discuss the critical role in molecular-dynamics modeling played by the angle (β) that individual C? H vectors make with the long axis of the DNA helix. Heat denaturation brings about increases in both the rate and amplitude of the internal motion (described by the wobble model with τ_W = ～0.2 × 10^?9 s, θ_cone = ～50°), and overall motion is affected by becoming essentially isotropic (τ_x = τ_Z = ～5 × 10^?8 s) for the single-stranded molecules. Since ¹³C-nmr data obtained at various DNA concentrations for C2′ of the deoxyribose ring is not described well by the above models, a new model incorporating an additional internal motion is proposed to take into account the rapid, extensive, and weakly coupled motion of C2′.     

Dna Damage by Chemically Generated Singlet Oxygen

A naphthalenic endoperoxide was used as a non-photochemical source of singlet oxygen (¹O₂) to examine some interactions between this reactive oxygen species and DNA. High molecular weight DNA (ca. 10⁸ daltons) was exposed to 120 mol m^?31O₂ (cumulative concentration) and analyzed for interstrand crosslinkage by hydroxyl apatite chromatography following formamide denaturation. No evidence for ¹O₂-induced interstrand crosslinking was obtained. The capacity of ¹O₂ to generate strand breaks in single-stranded (ss) and double-stranded (ds) DNA was investigated by sucrose gradient centrifugation analysis of bacteriophage øX174 DNA. No direct strand breaks could be detected at neutral pH, whereas extensive strand breakage was observed after treatment with alkali. Possible biological consequences of ¹O₂ -exposure were assessed by examining the plaque-forming capacity of ss and ds øX 174 DNA molecules using wildtype Escherichia coli spheroplasts as recipients. Without any further treatment with heat or alkali, exposure to the endoperoxide resulted in a time- and dose-dependent inactivation, ss DNA being considerably more sensitive than ds DNA. From the present results and those reported earlier (Nieuwint et al.,²⁰) we infer that ¹O₂-induced inactivation of øX174 DNA is not due to DNA backbone breakage nor to interstrand crosslinking, but rather to some form of damage to the base or sugar moiety of the DNA, the exact nature of which remains to be elucidated.     

The Effect of CO2 Enrichment on the Growth of Nodulated and Non-Nodulated Isogenic Types of Soybean Raised Under Two Nitrogen Concentrations

To find the effects of CO₂ enrichment on plant development and photosynthetic capacity of nodulated (line A62-1) and non-nodulated (line A62-2) isogenic lines of soybean (Glycine max Merr.), we examined the interactions among two CO₂ treatments (36±3 Pa = AC and 70±5 Pa = EC), and two nitrogen concentrations [0 g(N) m⁻²(land area) = 0N; 30 g(N) m⁻²(land area) = 30N]. Nodules were found in both CO₂ treatments in 0N of A62-1 where the number and dry mass of nodules increased from AC to EC. While the allocation of dry mass to root and shoot and the amount of N in each organ did not differ between the growth CO₂ concentrations, there was larger N allocation to roots in 0N than in 30N for A62-2. The CO₂-dependence of net photosynthetic rate (P _N) for A62-1 was unaffected by both CO₂ and N treatments. In contrast, the CO₂-dependence of P _N was lower in 0N than in 30N for A62-2, but it was independent of CO₂ treatment. P _N per unit N content was unaffected by CO₂ concentrations. The leaf area of both soybean lines grown in 30N increased in EC. But in 0N, only the nodulated A62-1 showed an increase in leaf area in EC. Nitrogen use efficiency of plants, NUE [(total dry mass of the plant)/(amount of N accumulated in the plant)] in 30N was unaffected by CO₂ treatments. In 0N, NUE in EC was lower than in AC in A62-1, and was higher than that at AC in A62-2. Hence, the larger amount and/or rate of N fixation with the increase of the sink-size of symbiotic microorganisms supplied adequate N to the plant under EC. In EC, N deficiency caused the down-regulation of the soybean plant. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interaction of poly(I)·poly(C) with trans-dichloro-diammine-platinum (II)

The covalent binding of trans-Pt (NH₃)₂Cl₂ to the double-stranded poly(I)·poly(C) follows three types of reactions, depending on r_b and the concentration of polynucleotide in the reaction mixture. At r_b ? 0.1, the principal reaction is coordination to poly(I), giving rise to some destabilization of the double strand, as shown by uv and CD spectra, and a decrease in T_m values, giving rise to free loops of poly(C). At higher r_b and low polynucleotide concentration, the free cytidine bases react with platinum bound on the complementary strand to form intramolecular (interstrand) crosslinks that restabilize the double-stranded structure. At high r_b and high polynucleotide concentration, while the above reaction still occurs, the predominant one is the formation of intermolecular crosslinks. Under no conditions has strand separation been observed.     

Kinetic study on unzippering of polynucleotides and order-order transition in polypeptides

Solutions are presented for N + 1 sequential and reversible first-order reactions for which the magnitude of the reverse rate constant, k_b, for all steps except the last is identical. Also the magnitude of the forward rate constant, k_f, for all steps except the first and last is identical. The initial and final steps are nucleation reactions; therefore, the initial and final k_f are modified by the factors σ′ and γ respectively. The final k_b is modified by the factor γ σ. The ratio k_b/k_f is defined as s, which has the same meaning as s in the Zimm-Bragg theory. The mathematical model is intended to apply to polymeric molecules of N segments and allows the calculation of the mole fraction of molecules in state i at any time t, C_i(t). A molecule in state i has i unreacted segments and N – i reacted ones. Because the reactions are sequential, all reacted segments are contiguous. Our numerical results show that when σ′ is much less than unity and the forward reaction is favored, the relaxation curve is sigmoidal. If, however, the forward and reverse reactions are equally favored (i.e., s ? 1) the relaxation curve is a straight line. When s and σ′ are near unity, the curve is exponential for a considerably large fraction of the reaction. Further, in the exponential for a considerably large fraction of the reaction. Further, in the exponential phase of the reaction, the relaxation time is proportional to N² for highly cooperative systems (i.e., Nσ ? 1). As found by Pipkin and Gibbs, if N is sufficiently large and s is less than unity (e.g., N ? 50 and s ?0.9) the relaxation curve is largely linear with a slope inversely proportional to N. Applications are given for the unwinding of double-helical poly(A·U) and the order–order transition in poly-L -proline.     

31P nmr spin-lattice relaxation studies of deoxyoligonucleotides

Temperature-dependent conformational transitions of deoxyoligonucleotides have been monitored by measuring ³¹P chemical shifts, spin-lattice relaxation times (T₁), and ³¹P-{H} nuclear Overhauser enhancements (NOEs). The measured NOE ranged from 30 to 80%, compared to the theoretical maximum of 124% for a dipolar relaxation mediated by rapid isotropic rotation. The observed 3′-5′ phosphate diester ³¹P T₁ showed a similar temperature dependence over the range 2–75°C for both double- and single-stranded oligonucleotides, and for dinucleotides. The results show that dipole–dipole interactions dominate the internucleotide phosphate relaxation rate in oligonucleotides. The same is true of terminal phosphate groups at low temperature; but at higher temperature another process, possibly due to contamination by paramagnetic ions, becomes dominant. The rotational correlation time τ_R calculated from the dipole–dipole relaxation rate of the internucleotide phosphate in d(pA)₂ at 16°C is τ_R = 5.0 × 10^?10 sec, implying a Stokes radius for isotropic rotation of 7.6 Å. The T₁ and NOE values for the double-helical octanucleotide d(pA)₃pGpC(pT)₃ are consistent with dominance of dipole–dipole relaxation and isotropic rotation of a sphere of radius 14 Å, a reasonable dimension for the double helix. Activation energies for the rotation of dinucleotides range from 4 to 6 kcal/mol, close to the value of 4 kcal/mol expected for isotropic rotation. In order to test the possible effect of internal motion of correlation time τ_G on the results, we considered a model in which the nucleotide chain rotates about the P-O bonds. Comparison of the calculation with our experimental results shows that internal motion with τ_G ? 10^?9 sec, as found from other studies to be present for large nucleic acids, would not influence out T₁ and NOE values enough to be distinguished from isotropic rotation. However, we can conclude that τ_G cannot be as fast as 10^?10 sec, even for dinucleotides.     

Comparative analysis of surface electrostatic potentials of carbon, boron/nitrogen and carbon/boron/nitrogen model nanotubes

We have extended an earlier study, in which we characterized in detail the electrostatic potentials on the inner and outer surfaces of a group of carbon and B_xN_x model nanotubes, to include several additional ones with smaller diameters plus a new category, C_2xB_xN_x. The statistical features of the surface potentials are presented and analyzed for a total of 19 tubes as well as fullerene and a small model graphene. The potentials on the surfaces of the carbon systems are relatively weak and rather bland; they are much stronger and more variable for the B_xN_x and C_2xB_xN_x. A qualitative correlation with free energies of solvation indicates that the latter two categories should have considerably greater water solubilities. The inner surfaces are generally more positive than the corresponding outer ones, while both positive and negative potentials are strengthened by increasing curvature. The outsides of B_xN_x tubes have characteristic patterns of alternating positive and negative regions, while the insides are strongly positive. In the closed C_2xB_xN_x systems, half of the C–C bonds are double-bond-like and have negative potentials above them; the adjacent rows of boron and nitrogens show the usual B_xN_x pattern. When the C_2xB_xN_x tubes are open, with hydrogens at the ends, the surface potentials are dominated by the B⁺–H^– and N^––H⁺ linkages.Figure Calculated electrostatic potential on the molecular surface of closed (6,0) B₄₈N₄₈; a is an outside view, while b shows the interior. Color ranges, in kcal mol^–1: red, greater than 20; yellow, between 20 and 0; green, between 0 and –10; blue, between –10 and –20; purple, more negative than –20     

Manipulation of single polymerase-DNA complexes: A mechanical view of DNA unwinding during replication

Comment on: Morin JA, et al. Proc Natl Acad Sci USA 2012; 109:8115-20.DNA replication requires overcoming the energetic barrier associated with the base pair melting of its double helix and a fine-tuned coordination between the processes of DNA unwinding and DNA replication. One intriguing question that remains poorly understood is the exact mechanism of the coupling of these two reactions. In some organisms, these activities are coupled within the same protein, like in the case of the phage Phi29 DNA polymerase. This polymerase works as a hybrid polymerase-helicase, because it presents an amino acid insertion that together with other protein domains forms a narrow tunnel around the template strand. This topological restriction is similar to the one imposed by hexameric helicases at the fork junction and promotes the separation of the fork ahead.¹ The Phi29 DNA polymerase, therefore, constitutes a simple, good model system to understand the basic mechanistic principles of the coupling between DNA replication and unwinding activities: the polymerase may behave as a “passive” unwinding motor, if translocation of the protein traps transient unwinding fluctuations of the fork, or as an “active” motor, if the polymerase actively destabilizes the duplex DNA at the junction. Therefore, factors that affect the stability of the fork junction, as DNA sequence or mechanical destabilization of the fork, will have a stronger effect on the unwinding kinetics of a “passive” motor than on an “active” one.To determine the DNA unwinding mechanism of the Phi29 DNA polymerase, we used optical tweezers to measure at single molecule level the effect of DNA sequence and destabilizing forces on the fork on the rates of strand displacement (replication and unwinding are tightly coupled, Δx₁, Fig. 1A) and primer extension (replication of the displaced complementary strand without unwinding, Δx₂, Fig. 1A) of two polymerases: the wild-type Phi29 DNA polymerase and a strand displacement deficient variant, which bears a couple of mutations that may affect the stability of the tunnel required for unwinding.² We quantified the free energy of interaction between the polymerase and the DNA fork, ΔG_int, and the range of this interaction, M, through a theoretical analysis of the dependence of the replication, unwinding and pause kinetics on the DNA sequence and force.^3,4Open in a separate windowFigure 1. (A) Schematic representation of the experimental design (not to scale). A single DNA hairpin was attached to functionalized beads inside a fluidics chamber. One strand of the hairpin is attached through a dsDNA handle to a bead held in the optical trap (top), while the complementary strand is attached to a bead on top of a mobile micropipette (bottom). At a constant force, after flowing the nucleotides into the reaction chamber, the strand displacement and primer extension rates of the polymerase are detected as a change in distance between the beads, Δx₁ and Δx₂, respectively. (B) Representative replication activity of a single mutant polymerase molecule. Long pauses are observed only during the strand displacement reaction. (C) Mechanistic distinction between passive and active unwinding. The cartoon illustrates the degree of activeness in DNA unwinding of different replicative helicases⁶ and the Phi29 DNA polymerase.Our results show that while the primer extension rates of both polymerases are force- and sequence-independent their average unwinding rates are sensitive to these two variables, although with different intensity. As expected, the dsDNA fork presents a much stronger physical barrier to the mutant polymerase unwinding. Qualitative reasoning might suggest that the observed differences imply different “activeness” of the unwinding mechanism of each polymerase. However, the inclusion of the pause kinetics of each polymerase in our model revealed that they use the same active mechanism; they both destabilize the two nearest base pairs of the fork (M = 2) with an interaction energy ΔG_int = 2 k_BT per base pair. These results suggest that mutations affecting the stability of the tunnel required for unwinding do not decrease the “activeness” of the motor but instead increase the probability of the unwinding mechanism to fail upon encountering a closed fork junction, inducing the entrance of the mutant polymerase into a long-lived inactive pause state (Fig. 1B). These results bring out the importance to consider pause kinetics to accurately quantify the actual unwinding mechanism of the Phi29 DNA polymerase or any other nucleic acid unwinding motor in which pauses are relevant during its operation. The presence of pauses obscures the actual pause-free rates of the motor and can lead to misleading results when they are not properly accounted.Our data are consistent with a model in which the closed template tunnel that wraps around the template strand allows the Phi29 DNA polymerase to maintain a sharp bending of this strand (essential for template reading in all replicative polymerases) and a bending of the complementary strand, due to its steric exclusion, at a closed fork junction. Bending of the two strands would generate mechanical stress at the junction promoting its active destabilization. A less stable tunnel, as in the mutant polymerase, will not be able to keep the mechanical stress at a closed fork junction, in this case the fork pressure would induce loosening of the correct protein-DNA interactions favoring the entrance to a polymerization inactive state.Similar mechanisms for mechanical destabilization of the fork junction can be envisioned for other DNA replication systems in which a DNA polymerase and a helicase work in coordination. In these systems, the leading strand can be sharply bent by the steric exclusion induced by the helicase and by the functional binding of the polymerase generating effective mechanical stress at the fork junction to account for efficient unwinding during replication. These implications are further supported by recent single molecule studies using magnetic tweezers that describe a collaborative coupling of this nature between the activities of the bacteriophage T4 DNA polymerase and DNA helicase.⁵     

Intramolecular and overall motion of proline: the influence of viscosity on carbon-13 spin-lattice relaxation times.

Nuclear magnetic resonance of ¹³C is used to probe the overall and internal motions of proline. Spin-lattice relaxation times (T₁) are reported for proline monomer dissolved in water/glycerol mixtures. Rates of overall molecular motion and internal motion depend on solvent composition but to different degrees. The effective correlation times (τ_eff) of the various proton-bearing carbon atoms in proline vary linearly as a function of solvent composition (%v/v) rather than of solution viscosity. The effective correlation time for molecular motion (τ_eff) is separated into contributions from overall molecular motion (τ_mol) and internal motion (τ_int). The γ-carbon of proline shows the smallest dependence of τ_int on solvent composition. The data indicate a high degree of intramolecular motion for the γ-carbon of proline. Inclusion of anisotropic molecular reorientation in the data analysis was found not to affect the above conclusions. The observed values of τ_eff indicate that the rotational diffusion model of molecular reorientations should apply to proline. The values of τ_eff calculated for proline using the Stokes-Einstein relation are larger than those observed; the discrepancy is discussed in terms of solvent-solute interactions.     

A comparison of 15N NMR relaxation measurements with a molecular dynamics simulation: Backbone dynamics of the glucocorticoid receptor DNA-binding domain

The rapid motions of the backbone of the DNA-binding domain of the glucocorticoid receptor (GR DBD) have been investigated using proton-detected heteronuclear NMR experiments on ¹⁵N-labeled protein at pH 6.0 and with a 200 psec molecular dynamics simulation of hydrated GR DBD. The experimental data were interpreted in terms of a generalized order parameter (S²) and an effective correlation time (τ_e) for the internal motion of each amide bond. A back calculation, using the same model, yielded the {¹H}-¹⁵N nuclear Overhauser effects (NOEs) and the ¹⁵N spin-lattice relaxation times (T₁) from the simulated data. The rapid motions of the backbone turned out to be rather limited and uniform throughout the protein, with a somewhat reduced mobility in the two major α-helical regions and a slightly enhanced flexibility for some residues in the first zinc coordinating region. The agreement between the experimental and simulated S²-values was as good as quantitative for most of the residues, except for some residues that were subject to a more large-scale, and in the simulation thus poorly sampled, motion. Examples of such motions that were found in the simulation include jumps of the amide bond of Ile-487 between the charged oxygens of the side chain of Asp-485 and less distinct large scale motions for some of the residues in the extended regions, that were shown to give rise to noisy and/or fast decaying internal reorientational correlation functions. For these residues large differences in the simulated and experimental τ_e-values were found, indicating that motions on different time scales were dominating in the experimental and simulated values. The lower (<0.7) experimental NOEs for these residues could not be reproduced in the simulation and were shown to be a consequence of the lower τ_e-values estimated in the simulation. By combining information from the simulation and the experiment a more complete picture of the motions for these residues can be obtained as is illustrated with an estimation of the jump angle and jump frequency for the amide bond of Ile-487. © 1993 Wiley-Liss, Inc.     

Characteristic effect of an anticancer dinuclear platinum(II) complex on the higher-order structure of DNA

It is known that a 1,2,3-triazolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH₃)₂}₂(μ-OH)(μ-1,2,3-ta-N ¹,N ²)](NO₃)₂ (AMTA), shows high in vitro cytotoxicity against several human tumor cell lines and circumvents cross-resistance to cisplatin. In the present study, we examined a dose- and time-dependent effect of AMTA on the higher-order structure of a large DNA, T4 phage DNA (166 kbp), by adapting single-molecule observation with fluorescence microscopy. It was found that AMTA induces the shrinking of DNA into a compact state with a much higher potency than cisplatin. From a quantitative analysis of the Brownian motion of individual DNA molecules in solution, it became clear that the density of a DNA segment in the compact state is about 2,000 times greater than that in the absence of AMTA. Circular dichroism spectra suggested that AMTA causes a transition from the B to the C form in the secondary structure of DNA, which is characterized by fast and slow processes. Electrophoretic measurements indicated that the binding of AMTA to supercoiled DNA induces unwinding of the double helix. Our results indicate that AMTA acts on DNA through both electrostatic interaction and coordination binding; the former causes a fast change in the secondary structure from the B to the C form, whereas the latter promotes shrinking in the higher-order structure as a relatively slow kinetic process. The shrinking effect of AMTA on DNA is attributable to the possible increase in the number of bridges along a DNA molecule. It is concluded that AMTA interacts with DNA in a manner markedly different from that of cisplatin.     

Thermodynamics of DNA Containing Very Stable Chemically Modified Base Pairs

Abstract

DNA chemical modifications caused by the binding of some antitumor drugs give rise to a very strong local stabilization of the double helix. These sites melt at a temperature that is well above the melting temperatures of ordinary AT and GC base pairs. In this work we have examined the melting behavior of DNA containing very stable sites. Analytical expressions were derived and used to evaluate the thermodynamic properties of homopolymers DNA with several different distributions of stable sites. The results were extended to DNA with a heterogeneous sequence of AT and GC base pairs. The results were compared to the melting properties of DNA with ordinary covalent interstrand cross-links. It was found that, as with an ordinary interstrand cross-link, a single strongly stabilized site makes a DNA's melting temperature (T_m ) independent of strand concentration. However in contrast to a DNA with an interstrand cross-link, a strongly stabilized site makes the DNA's T_m independent of DNA length and equal to T _∞, the melting temperature of an infinite length DNA with the same GC-content and without a stabilized site. Moreover, at a temperature where more than 80% of base pairs are melted, the number of ordinary (non-modified) helical base pairs (n) is independent of both the DNA length and the location of the stabilized sites. For this condition, n(T) = (2ω-a) S (1- S ) and S = exp[ΔS(T∞-T)/(RT)] where ω is the number of strongly stabilized sites in the DNA chain, a is the number of DNA ends that contain a stabilized site, and ΔS, T, and R are the base pair entropy change, the temperature, and the universal gas constant per mole. The above expression is valid for a temperature interval that corresponds to n<0.2N for ω=1, and n<0.1N for ω>1, where N is the number of ordinary base pairs in the DNA chain.     

Walking of antitumor bifunctional trinuclear PtII complex on double-helical DNA

The trinuclear BBR3464 ([{trans-PtCl(NH₃)₂}₂µ-(trans-Pt(NH₃)₂(H₂N(CH₂)₆NH₂)₂)]⁴⁺) belongs to the polynuclear class of platinum-based anticancer agents. DNA adducts of this complex differ significantly in structure and type from those of clinically used mononuclear platinum complexes, especially, long-range (Pt, Pt) intrastrand and interstrand cross-links are formed in both 5′–5′ and 3′–3′ orientations. We show employing short oligonucleotide duplexes containing single, site-specific cross-links of BBR3464 and gel electrophoresis that in contrast to major DNA adducts of clinically used platinum complexes, under physiological conditions the coordination bonds between platinum and N7 of G residues involved in the cross-links of BBR3464 can be cleaved. This cleavage may lead to the linkage isomerization reactions between this metallodrug and double-helical DNA. Differential scanning calorimetry of duplexes containing single, site-specific cross-links of BBR3464 reveals that one of the driving forces that leads to the lability of DNA cross-links of this metallodrug is a difference between the thermodynamic destabilization induced by the cross-link and by the adduct into which it could isomerize. The rearrangements may proceed in the way that cross-links originally formed in one strand of DNA can spontaneously translocate from one DNA strand to its complementary counterpart, which may evoke walking of the platinum complex on DNA molecule.     

Sedimentation coefficients of DNA topoisomers: the helical wormlike chain

The sedimentation coefficient s_N of the DNA topoisomer with the linking number N is evaluated as a function of N and chain length on the basis of a (circular) twisted wormlike chain, i.e., a special case of the helical wormlike chain. Evaluation is carried out by an application of the Oseen–Burgers procedure of hydrodynamics to the cylinder model with the preaveraged Oseen tensor. The necessary mean reciprocal distance between two contour points is obtained by a Monte Carlo method. It is shown that s_N increases as |ΔN| is increased from 0 in the range of small |ΔN|, where ΔN = N ? N , with N the number of helix turns in the linear DNA chain in the undeformed state. It is found that there is semiquantitative agreement between the Monte Carlo values and the experimental data obtained by Wang for s_N.     

Photon correlation spectroscopy, total intensity light scattering with laser radiation, and hydrodynamic studies of a well fractionated DNA sample.

A Malvern laser light-scattering instrument has been modified for use at scattering angles down to 5° and both total intensity and quasi-elastic scattering experiments. A sample of sheared, length-fractionated calf-thymus DNA was characterized by sedimentation, viscosity and electron microscopy. Quasi-elastic scattering and absolute intensity determinations were performed with the laser instrument and intensity determinations only with a Fica conventional light-scattering photometer. The total intensity experiments gave M?_w = (3.75 ± 0.15) × 10⁶ and 〈R²〉^1/2_z = (206.9 ± 10.3) nm which yielded a value for the persistence length, allowing for polydispersity, of 66 ± 6nm. The quasi-elastic experiments at scattering angles below 20° gave D⁰_{20, w} = (2.23 ± 0.06) × 10^?8 cm²/sec which combined with S⁰_{20, w} = 15.6 in the Svedberg equation gave M?_w = (3.73 ± 0.18) × 10⁶. In addition, from the higher angle data we extracted a value of the longest intramolecular relaxation time, τ1 of 17.5 msec. This is not in particularly good agreement with τ1 predicted by the Zimm–Rouse theory using our other experimental parameters. The disagreement may be due to the restricted applicability of the Zimm–Rouse spring-bead model as a quantitative representation of DNA molecules. Alternatively, it may be due to present difficulties in the unambiguous interpretation of molecular motions from the experimental autocorrelation functions.     

DNA binding of dinuclear iron(II) metallosupramolecular cylinders. DNA unwinding and sequence preference

[Fe₂L₃]⁴⁺ (L = C₂₅H₂₀N₄) is a synthetic tetracationic supramolecular cylinder (with a triple helical architecture) that targets the major groove of DNA and can bind to DNA Y-shaped junctions. To explore the DNA-binding mode of [Fe₂L₃]⁴⁺, we examine herein the interactions of pure enantiomers of this cylinder with DNA by biochemical and molecular biology methods. The results have revealed that, in addition to the previously reported bending of DNA, the enantiomers extensively unwind DNA, with the M enantiomer being the more efficient at unwinding, and exhibit preferential binding to regular alternating purine–pyrimidine sequences, with the M enantiomer showing a greater preference. Also, interestingly, the DNA binding of bulky cylinders [Fe₂(L-CF₃)₃]⁴⁺ and [Fe₂(L-Ph)₃]⁴⁺ results in no DNA unwinding and also no sequence preference of their DNA binding was observed. The observation of sequence-preference in the binding of these supramolecular cylinders suggests that a concept based on the use of metallosupramolecular cylinders might result in molecular designs that recognize the genetic code in a sequence-dependent manner with a potential ability to affect the processing of the genetic code.     

Molecular-Mechanical studies of the mismatched base analogs of d(CGCGAATTCGCG)2:d(CGTGAATTCGCG)2, d(CGAGAATTCGCG)2, d(CGCGAATTCACG)2, d(CGCGAATTCTCG)2, and d(CGCAGAATTCGCG)·d(CGCGAATTCGCG)

Molecular-mechanical simulations have been carried out on “mismatched base” analogs of the DNA double-helical structure d(CGCGAATTCGCG)₂, in which the base pairs CG at the 3 and 10 positions have been replaced by CA, AG, TC, and TG base pairs, as well as an insertion analog in which an extra adenine has been incorporated into one strand of the above structure between bases 3 and 4. The results of these simulations (calculated relative stabilities, structures, and nmr ring-current shifts) have been compared with calorimetric and nmr data. The calculated relative stabilities of the double-helical parent dodecamer and the various “wobble” base pairs qualitatively correlate with the experimental melting temperatures. The base-pairing structure for the GT wobble pair is in agreement with that previously determined from nmr experiments. For the GA base pair, the structure with both bases anti has a slightly more favorable energy from base pairing and stacking than a structure with non-Watson-Crick H-bonding with adenine syn, in agreement with nmr experiments. The CA wobble base is calculated to favor an adenine 6NH₂ …? cytosine N3 H-bond over cytosine 4NH₂ …? adenine N1, again, in agreement with nmr experiments. There is no definitive experimental data on the TC base pair, but the existence of (somewhat long and weak) H-bonds involving cytosine 4NH₂ …? thymine 4CO and cytosine N3 …? thymine HN3 seems reasonable. We find a structure in which the extra adenine base of the insertion analogs sits “inside” the double helix.     

The emergence of molecular machines as a prerequisite of the ancient RNA world evolution

The problem of the start of biological evolution in the ancient RNA world is considered. It is postulated that the appearance of catalytic RNAs — ribozymes — via spontaneous cis- and trans-rearrangements of polyribonucleotides in primordial Darwin ponds should not have been sufficient for the start of evolution, until a new class of functional RNA, namely energy-dependent molecular machines, arose. The proposed hypothesis is that the simplest and primary type of molecular machines could be nucleoside triphosphate-dependent RNA-based helicases, which were capable of unwinding the stable double-helical RNAs inevitably formed during RNA syntheses on complementary templates. Thereupon, unwinding RNA polymerases could appear as a result of association or fusion of helicases and polyribonucleotide-polymerizing ribozymes. The latter event provided the mechanism of RNA replication using the double-helical RNAs as a communal genofond (gene pool) of a Darwin pond, and thus initiated the fast evolution of the ancient RNA world.