Kinetics of the helix-coil transition in DNA

The kinetics of the helix-coil transition have been investigated for T2 and T7 phage DNA in a formamide-water-salt mixed solvent using a slow temperature perturbation technique (applicable to kinetic processes with rate constants ? 3 min^?1). In this solvent degradation of the DNA is effectively suppressed. Complex kinetic curves are observed by absorbance and viscosity measurements for the response to denaturing perturbations in the transition region. Analysis of the decay curves indicates that the denaturation reaction in this time range can be treated as a first-order reaction with a variable first-order rate parameter, k, the derivative of the logarithm of the absorbance or viscosity change with respect to time. In the approach to denaturation equilibrium in the transition region, the rate parameter is determined only by the instantaneous extent of denaturation of the molecules. Near equilibrium, the rate parameter assumes a constant value characteristic of the equilibrium state. In this region, where the denaturation reaction proceeds as a simple first-order process, both the decay of absorbance (reflected local conformational change) and the decay of solution viscosity (reflecting macromolecular conformational change) are characterized by the same constant value of k. In 83% formamide, 0.3M Na⁺, the rate parameter k for T2 DNA decreases from an extrapolated value of 2.0 min^?1 at 0% denaturation to 0.11 min^?1 at 90% denaturation. Rate parameters determined for T7 DNA at the same counterion concentration and fraction of denaturation are approximately five times as large as those cited for T2 DNA, indicating an inverse proportionality of rate constant to molecular length. On the other hand, simple first-order kinetic responses with constant k are obtained for renaturing perturbations within the transition, indicating that the mechanism of rewinding differs, in most cases, from that of unwinding. Only in the limit of very small perturbations about a given equilibrium position are the rate constants k obtained from denaturing and renaturing perturbations equal. For perturbations of finite size, it appears possible that an intramolecular initiation or nucleation event may precede rewinding and limit the rate of this reaction. The rate parameters again are approximately inversely proportional to molecular weight. The one exception to the first-power dependence on molecular weight appears when temperature jumps are made upward into the post-transition region. Here the molecular-weight dependence is second power, but complications arising from the different strand-separation properties of T2 and T7 DNA's make interpretation difficult. The previously used model of friction-limited unwinding appears to fit all the observations except for the molecular-weight dependence.     

Kinetics of denaturation of DNA

The kinetics of denaturation of DNA have been studied by relaxation techniques. Examination of the terminal relaxation times for a variety of DNA's under a variety of conditions has shown that DNA denaturation is principally a hydrodynamically limited process. Measurements within the helix–coil transition have demonstrated that the experimentally measured terminal relaxation times are a function of the following: (1) position in the helix–coil transition; (2) ionic strength of the solvent; (3) solvent viscosity; (4) DNA concentration; (5) molecular weight; (6) number and position of single-strand breaks. The dependence of the terminal relaxation time on the above mentioned factors can be attributed to hydrodynamic effects. Thus a hydrodynamic model for DNA unwinding is required. The model which best fits the data involves the assumption of a rotational frictional coefficient independent of molecular weight. This assumption is suggested by the fact that the relaxation time is proportional to the first power of the molecular weight.     

DNA denaturation kinetics in CHO cells exposed to different X-ray doses and after different repair intervals using the alkaline unwinding technique

Summary The kinetics of DNA denaturation in alkaline solution (pH 12.2) was studied in CHO cells using the alkaline unwinding technique. After X-ray doses of 0, 3, 5 and 9 Gy, the kinetics of alkaline denaturation was found to be independent of the number of induced strand breaks confirming earlier studies on this subject. In addition, the denaturation kinetics measured in cells exposed to 9 Gy were found to be identical for different repair intervals. This result shows that for the three different classes of DNA strand breaks described previously (Dikomey and Franzke 1986a) strand separation in alkaline solution occurs at the same kinetics. As a consequence, the relationship between the numbers of strand breaks and the fraction of remaining double-stranded DNA is considered the same for the three different classes.     

Theoretical and Experimental Study of DNA Helix-Coil Transition in Acidic and Alkaline Medium

Abstract

The theoretical approach to the calculation of the influence of selective binding of small ligands on DNA helix-coil transition has been described in the previous paper (Lando D.Yu., J. Biomol. Struct. Dyrt., (1994)). In the present paper that method is used for the study of DNA protonation and deprotonation in acidic and alkaline medium by theoretical analysis of pH effect on DNA heat denaturation.

The mechanism of DNA protonation in acidic medium and pK values of nucleotides are well known. It gave us an opportunity to check the theory without any fitting of pK values. A good agreement between experimental and calculated functions T_m(pH) and ΔT(pH) (melting temperature and melting range width) obtained for acidic medium proved the validity of the theory. However, for alkaline medium there was not even qualitative agreement when the agreed-upon mechanism of deprotonation was considered. Looking into the cause of the discrepancy, we have studied the DNA melting for different mechanisms of deprotonation by calculation of T_m(pH) and ΔT(pH). As a result, it has been established that the discrepancy is due to deprotonation of bonded GC base pairs of helical DNA regions (pK= 11).

It was shown that the early known protonation and newly found deprotonation of helical DNA essentially stabilised double helix in alkaline and acidic medium.     

Association-dissociation and denaturation behaviour of an oligomeric seed protein alpha-globulin of Sesamum indicum L. in acid and alkaline solutions.

The association-dissociation and denaturation behaviour of the major protein fraction, alpha-globulin of sesame seed (Sesamum indicum L.), in acid and alkaline solutions in the ranges of pH 4.2-1.5 and pH 7-12 have been studied. The results of gel filtration, fluorescence and viscosity measurements indicate dissociation and denaturation of the protein up to pH approximately 3. The difference spectrum in this region arises from a combination of dissociation, denaturation and charge effect on the chromophore. In still stronger acid solution, reassociation of the dissociated fraction takes place by hydrophobic interaction. In alkaline solution dissociation takes place around pH 8, and above pH 10 dissociation and denaturation proceed simultaneously as has been evidenced by sedimentation, fluorescence, spectral change, optical rotation and viscosity measurements. The phenolic group (pKInt=10.6) in the protein is abnormal and denaturation in alkaline solution is irreversible. Above pH 11.5 further dissociation of the protein takes place. Characteristic pH values of transition from 10.6-10.8 indicate that the transition of the protein involves a single step in alkaline solution.     

Studies on conformational changes in the DNA structure induced by protonation: reversible and irreversible acid titrations and sedimentation measurments

Reversible and irreversible conformational changes in the acid-induced denaturation of DNA were studied by spectrophotometric titration, sedimentation, and melting measurements. A GC-rich DNA (72 mole-%) shows complete or partial reversibility of the titration profiles within the pH region of transition from helix to coil, while AT-rich DNA (29 mole-%) is irreversible in its titration behavior at each acid pH below the onset of the transition. The results for GC-rich DNA further indicate distinct differences in the titration behavior, which can be attributed to differences in the frequency of GC clusters along the DNA molecule. Plots of the sedimentation coefficient and the parameter a_s^app against pH lead to the conclusion that conformational changes occur before the onset of the acid-induced helix–coil transition. These alterations are more pronounced upon protonation of larger GC-rich domains than of smaller ones, as concluded from very marked differences observed in the sedimentation–pH behavior of two GC-rich DNA's. An acid denaturation scheme for a GC-rich DNA segment is suggested. Reversibility of the acid denaturation is explained by the existence of stable, protonated, single GC base pairs in nonprotonated stacked single-stranded domains formed in the acid-induced transition region.     

Thermodynamic and structural study of phenanthroline derivative ruthenium complex/DNA interactions: probing partial intercalation and binding properties

The binding of [Ru(PDTA-H₂)(phen)]Cl (PDTA = propylene-1,2-diaminetetra-acetic acid; phen = 1,10 phenanthroline) with ctDNA (=calf thymus DNA) has been investigated through intrinsic and induced circular dichroism, UV-visible absorption and fluorescence spectroscopies, steady-state fluorescence, thermal denaturation technique, viscosity and electrochemical measurements. The latter indicate that the cathodic and anodic peak potentials of the ruthenium complex shift to more positive values on increasing the DNA concentration, this behavior being a direct consequence of the interaction of both the reduced and oxidized form with DNA binding. From spectrophotometric titration experiments, the equilibrium binding constant and the number of monomer units of the polymer involved in the binding of one ruthenium molecule (site size) have been quantified. The intrinsic circular dichroism (CD) spectra show an unwinding and a conformational change of the DNA helix upon interaction of the ruthenium complex. Quenching process, thermal denaturation experiments and induced circular dichroism (ICD) are consistent with a partial intercalative binding mode.     

Radiation-induced DNA unwinding is influenced by cell shape and trypsin.

Incubation of cells in high salt/alkali typically leads to denaturation and unwinding of DNA, yet DNA from Chinese hamster V79 cells grown for 1 day as spheroids stops unwinding after only 5-10 min. We previously postulated that this was a result of "constraints" to DNA unwinding present in cells in spheroids but not in monolayers, and that these constraints could be responsible for the increased resistance of spheroids of V79 cells to killing by ionizing radiation (i.e., the contact effect). However, studies reported here indicate that this limited DNA unwinding is correlated with a round cell shape and lack of cell surface fibronectin. In round cells which continue to synthesize fibronectin, demonstration of constraints requires prior exposure to trypsin in order to digest cell surface fibronectin. However, trypsin did not influence cell killing by ionizing radiation. Therefore, the increase in radiation resistance of V79 spheroids and the change in their DNA unwinding kinetics both appear contingent upon a change in cell shape; differences in DNA denaturation rates which are detected in spheroids using the unwinding assay are apparently not directly responsible for the contact effect.     

Formation of strand breaks and interstrand cross-links in DNA by methylglyoxal

Methylglyoxal (MG), a dietary mutagen, is present in various frequently consumed beverages and foods and in cigarette smoke. A combination of S1 nuclease hydrolysis and alkaline unwinding assay was used to demonstrate the formation of single-strand breaks and interstrand cross-links in DNA upon treatment with MG. Calf thymus DNA, when treated with increasing concentrations of MG, showed an increasing degree of S1 nuclease hydrolysis. It also showed the formation of an increasing number of strand breaks per molecule as determined by an alkaline unwinding assay. Incubation of DNA with relatively higher concentrations of methylglyoxal or prolonged treatment gave increased thermal melting temperatures and an enhanced rate of reannealing after thermal denaturation. These results indicated the formation of interstrand cross-links. Upon treatment with MG, A-T base pair depleted DNA showed a reduced number of single-strand break formation. It also showed a significantly lower decrease in Tm as compared with MG-treated normal DNA. These results showed that under the conditions used, MG primarily reacts with A-T base pairs in duplex DNA.     

Sedimentation and intrinsic viscosity behavior of PM2 bacteriophage DNA in alkaline solution

The sedimentation coefficient and intrinsic viscosity of nicked and closed circular PM2 bacteriophage DNA have been measured as a function of pH in the alkaline region. A gradual increase in the sidimentation coefficient, and a corresponding decrease in the intrinsic viscosity, are observed for the superhelical (closed) circle in the pH region from 10.5 to about 10.9. This has been tentatively interpreted in terms of the known dependence of sedimentation coefficient upon the number of superhelical turns. At slightly higher pH values, the curve passes through the minimum (sedimentation coefficient) and maximum (intrinsic viscosity) expected when the superhelical turns present at neutral pH are unwound by partial alkaline denaturation. Sedimentation studies of the relaxed (nicked) circular species have revealed the existence of DNA forms in the pH region from 11.27 to 11.37 which sediment considerably faster than the closed circle in the same pH region. These have been identified as partially denatured nicked circles, in which varying fractions of the duplex structure have undergone alkaline denaturation, but strand separation has not yet occurred. Varying fractions of a slower species, either undenatured or completely denatured nicked circles, are also observed in some of these experiments. A corresponding result is observed in the intrinsic viscosity vs. pH curve. When nicked circular PM2 DNA is exposed to various alkaline pH's, rapidly neutralized, and sedimented at neutral pH, the expected sharp transition from native to denatured (strand-separated) molecules is seen. However, a very narrow pH range is noted in which native and denatured forms coexist in a single experiment. The above experiments carried out upon the closed form also reveal a narrow pH range in which the bulk of the transition from native closed circles to the collapsed cyclic coil takes place, in acccord with an earlier study on a different DNA. This transition is shown never to be completely effected, however, as there is a fraction (7–8%)of the closed circles which renature to the native form, regardless of the alkaline pH employed. This same phenomenon was not observed in the case of artificially closed λb₂b₅c DNA circles. Possible explanations for some of the above results are discussed.     

Reversibility of thermal transitions in proteins: Racemization and aggregation as factors in the reversible denaturation of a soluble keratin derivative (SCMKA)

The reversibility of the thermal denaturation of a low-sulfur fraction of solubilized wool keratin (SCMKA) has been studied under a variety of conditions of time, protein concentration, and pH. Two types of irreversibility for the transition have been encountered. One of these is associated with an aggregation of the protein on denaturation to give a product which may contain elements of a β conformation. This type of irreversibility is favored by high protein concentration, and the original conformation may in fact be regained if the aggregated structure is broken down by a solvent such as 8M urea and the urea subsequently removed by dialysis. The other type of irreversibility appears to be due to racemization of the protein. It does not seem to be dependent on protein concentration and is apparent only at temperatures beyond the actual transition range (～40–65°C) at pH values below 11, At pH 12, however, racemization appears to proceed slowly even at 4°C. The thermal transition at pH 9 and pH 10 has been shown to be multistage in nature. Over the pH range 9–12 there is a progressive decrease in thermal stability with increase of pH. Addition of NaCl at pH 10 leads to an increase in thermal stability of the molecule.     

Characteristics of M-Gtfi,A New Inhibitor of Streptococcus Mutans Glucosyltransferase

Abstract

M-GTFI, originally screened as an inhibitor of Streptococcus mutans glucosyltransferase, strongly inhibited α-glucosidase, in a non-competitive manner especially when the synthetic substrate p-nitrophenyl-α-D-glucopyranoside was used. It also inhibited β-glucosidase, β-amylase and, to a lesser extent, β-glu-curonidase.

The inhibitor was stable in neutral and alkaline pH ranges and dependency of the inhibition on pH and temperature was not observed. Some proteinases and polysaccharides-hydrolyzing enzymes as well as human saliva did not inactivate the inhibitor.

There was a correlation between the release of sulfate anions from the inhibitor molecule on incubation with HCI (0.2 N) at 100°c and loss of inhibitory properties of the molecule. It is suggested that the presence of sulfate ester linkages in the inhibitor molecule play an important role in the inhibition process.     

A hydroxylapatite batch assay for quantitation of cellular DNA damage.

A batch elution procedure is described for quantitative measurement of DNA damage. The technique is based on alkaline unwinding of cellular DNA and separation of singlestranded from duplex forms by step elution from hydroxylapatite with phosphate formamide. The method is rapid, permits large numbers of samples to be handled simultaneously, and consistently yields recoveries of >95% of total chromatographed DNA. Because as many as 1 × 10⁷ cells per batch may be analyzed, quantitation of the eluted DNA by nonradioactive methods is feasible. The method is standardized with respect to the unwinding constant β, the alkaline DNA unwinding unit Mng, and the DNA-damaging efficiency of ionizing irradiation.     

Detection of DNA damage induced in vivo by a cross-linking agent with a circular channel crucible oscillating viscometer

DNA damage induced in vivo by the cross-linking agent mitomycin C (MMC) was investigated with a new oscillating crucible viscometer. Viscosity was measured by lysing rat liver nuclei in an alkaline lysing solution (pH 12.5; 25 degrees C). In control samples the viscosity increased very slowly with time, reaching a plateau only after 10-12 h. The process was accelerated and the maximum viscosity was decreased by alkaline single-stranded breaks arising from methylation and subsequent depurination of DNA in vitro with dimethylsulphate (DMS). MMC, when given alone, had no evident effect on the time needed for reaching plateau viscosity but it induced a small increase in maximum viscosity. When MMC was given in association with DMS, the time of disentanglement remained unchanged (accelerated) but maximum viscosity was increased in a dose dependent way. We conclude that these data clearly confirm that the slow steady increase of the viscosity of control DNA with time reflects mainly the process of unwinding of the two strands. The speed of this process seems to depend only from the number of unwinding points in DNA (breaks).     

Structural analysis and molecular modeling of the RvH2-e functional unit of Rapana venosa hemocyanin

Rapana venosa hemocyanin (RvH), a circulating glycoprotein of the marine snail, has a complex structure. To provide details on the stability of the protein, one functional unit, RvH2-e, was compared with the native molecule and the structural subunits, RvH1 and RvH2, via pH–T diagrams, typical phase portraits for stability and denaturation reversibility. By analyzing the T transition curves of RvH2-e at different pH values, several parameters of the thermodynamic functions were obtained. Increasing the temperature from 25 °C to 55 °C, the reversibility of the molecule of protein also increases, opening a reversibility window within the range of pH 4.0–8.0. On analyzing the pH transition curves, the start of the acid denaturation (below pH 6) and alkaline denaturation (above pH 9) was determined to be between 20 °C and 35 °C. For this range, the thermodynamic functions ΔH° and ΔG° for a standard temperature of 25 °C were calculated.     

A rapid method for detecting DNA strand breaks in Mytilus galloprovincialis Lam. induced by genotoxic xenobiotic chemicals

1. In this study, DNA from haemolymph cells of Mytilus galloprovincialis Lam., as well as from L1210 (murine leukemia) mouse cells was investigated utilizing the technique of the alkaline unwinding of the double stranded DNA molecule. 2. The data show that DNA of haemolymph cells from the marine invertebrate has an unwinding time and, therefore, a molecular weight considerably lower than that of DNA of mammalian cells. 3. The exposure of the cells from mussel haemolymph and from mouse L1210 to a genotoxic compound such as dimethylsulfate results in DNA damage and consequently in a reduction of the unwinding time. 4. These results suggest that the fluorimetric DNA unwinding assay can be used in studies concerning the damage of DNA of marine organisms induced by genotoxic compounds or environmental factors.     

The binding of echinomycin to deoxyribonucleic acid.

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.     

Spectroscopic studies on proteinase K and subtilisin DY. Relation to X-ray models.

Circular dichroic spectroscopy has been used to study the effect of pH, guanidinium hydrochloride concentration and temperature on the conformation of the fungal subtilisin-like proteinase K and the bacterial DY. The ellipticity of the bands in the far ultraviolet region remains almost unchanged in the pH range 3.0-11.0 (PMS-proteinase K) and 5.0-10.0 (PMS-subtilisin DY). The same ranges of pH stability were determined from the pH dependence of the near ultraviolet dichroic spectra. Hence the changes in the tertiary and secondary structure occur in parallel. Proteinase K is considerably more stable at acidic and somewhat more stable at alkaline pH than subtilisin DY. At neutral pH proteinase K is more resistant to denaturation by guanidinium hydrochloride than is subtilisin DY. The midpoints of the denaturation curves were 6.2 M and 3.2 M guanidinium, respectively. The thermal unfolding of proteinase K occurred at a higher temperature than for subtilisin DY, the transition midpoints being 65 degrees and 48 degrees, respectively. Thus proteinase K is overall a much more robust molecule than subtilisin DY, showing greater resistance to all three forms of denaturation. The differences in the stability of the two proteinases can be partly explained by differences in their calcium binding sites.     

The degree of unwinding of the DNA helix by ethidium. I. Titration of twisted PM2 DNA molecules in alkaline cesium chloride density gradients

A series of covalently closed bacteriophage PM2 DNA samples with varying degrees of superhelicity were prepared in vitro. The amount of bound ethidium per DNA nueleotide needed for the removal of all superhelical turns, v_c⁰, was determined for each sample by a number of methods. In order to evaluate the unwinding angle for the binding of one ethidium molecule to a DNA double helix, the pH dependence of the buoyant densities in CsCI of these samples was examined. A new calibration relating the change in buoyant density of a DNA to the fraction of bases titrated has been obtained, by measuring the buoyant densities of a number of catenanes (interlocked rings) containing both single-stranded and double-stranded λ DNA rings, at a pH such that the single-stranded DNA is fully titrated while the double-stranded DNA is not titrated. This calibration was used to obtain the pH dependence of the fraction of DNA bases titrated for the phage PM2 DNAs with differing extents of supercoiling. A simple theoretical analysis shows that in a restricted pH range close to pH_m, the melting pH of the DNA in the absence of the topological constraint associated with covalently closed double-stranded DNAs, the difference in the fraction of bases titrated at a certain pH between two covalently closed DNAs with different degrees of superhelicity is directly proportional to the difference in the v_c⁰ values of the DNAs. The unwinding angle per bound ethidium molecule can be obtained from the proportionality constant. In this way, it is not necessary to know precisely the actual pH value for either DNA, pH_e, at which the DNA is titrated to the extent that it contains no superhelical turns. The conclusion of the theoretical analysis and the experimental results is that the binding of an ethidium molecule to a double-stranded DNA unwinds the DNA helix by an angle φ_e = 26 °. The uncertainty in this value is estimated to be less than 10%. The new value for φ_e is approximately a factor of two larger than the value 12 °, which has been in use in the past decade. In the earlier alkaline titration results for polyoma DNA (Vinograd et al., 1968), which had been interpreted as supporting the 12 ° value, the calculation of φ_e was critically dependent on knowing pH_e. It is believed that pH_e was underestimated in the earlier work, resulting in a low φ_e value. Since the previous value φ_e = 12 ° has been widely used in the determination of the number of superhelical turns for many DNAs, and in measurements on the angular alterations of the DNA helix by the binding of a variety of small and large molecules and by solvent and temperature changes, the new value φ_e = 26 ° requires proportional adjustments of many previous results.     

Repair of DNA single-strand breaks in X-irradiated yeast. I. Use of the DNA-unwinding method to measure DNA strand breaks

The DNA-unwinding method developed by Ahnstr?m and his coworkers to measure DNA strand breaks in mammalian cells was used to measure single-strand breaks (SSB) in the DNA of intact yeast cells. DNA unwinding, which took place inside the rigid cell wall of yeast, was investigated as a function of time, radiation dose, and of pH and salt concentration of the alkaline solution. After DNA unwinding had taken place, the cell wall was destroyed by partial enzymatic digestion and sonication in the presence of detergents. Fragments of single- and double-stranded DNA were separated using hydroxylapatite chromatography. In this way the most suitable conditions for DNA unwinding within the cell wall were established. The results show that SSB and double-strand breaks (DSB) give rise to different kinetics of DNA unwinding.