Transfection of Escherichia coli Spheroplasts II. Relative Infectivity of Native, Denatured, and Renatured Lambda, T7, T5, T4, and P22 Bacteriophage DNAs

The change of infectivity of phage DNAs after heat and alkali denaturation (and renaturation) was measured. T7 phage DNA infectivity increased 4- to 20-fold after denaturation and decreased to the native level after renaturation. Both the heavy and the light single strand of T7 phage DNA were about five times as infective as native T7 DNA. T4 and P22 phage DNA infectivity increased 4- to 20-fold after denaturation and increased another 10- to 20-fold after renaturation. These data, combined with other authors' results on the relative infectivity of various forms of phiX174 and lambda DNAs give the following consistent pattern of relative infectivity. Covalently closed circular double-stranded DNA, nicked circular double-stranded DNA, and double-stranded DNA with cohesive ends are all equally infective and also most highly infectious for Escherichia coli lysozyme-EDTA spheroplasts; linear or circular single-stranded DNAs are about 1/5 to 1/20 as infective; double-stranded DNAs are only 1/100 as infective. Two exceptions to this pattern were noted: lambda phage DNA lost more than 99% of its infectivity after alkaline denaturation; this infectivity could be fully recovered after renaturation. This behavior can be explained by the special role of the cohesive ends of the phage DNA. T5 phage DNA sometimes showed a transient increase in infectivity at temperatures below the completion of the hyperchròmic shift; at higher temperatures, the infectivity was completely destroyed. T5 DNA denatured in alkali lost more than 99.9% of its infectivity; upon renaturation, infectivity was sometimes recovered. This behavior is interpreted in terms of the model of T5 phage DNA structure proposed by Bujard (1969). The results of the denaturation and renaturation experiments show higher efficiencies of transfection for the following phage DNAs (free of single-strand breaks): T4 renatured DNA at 10(-3) instead of 10(-5) for native DNA; renatured P22 DNA at 3 x 10(-7) instead of 3 x 10(-9) for native DNA; and denatured T7 DNA at 3 x 10(-6) instead of 3 x 10(-7) for native DNA.     

Helix-coil transition in nucleoprotein: renaturation of polylysine-DNA and polylysine-nucleohistone complexes

Thermal denaturation and renaturation of directly mixed and reconstituted polylysine–DNA, directly mixed polylysine–nucleohistone complexes, and NaCl-treated nucleohistones in 2.5 × 10^?4 M EDTA, pH 8.0 have been studied. At the same input ratio of polylysine to DNA, the percent of renaturation of free base pairs in a directly mixed polylysine–DNA complex is higher than that in a reconstituted complex. For a directly mixed complex, the renaturation of free base pairs is proportional to the fraction of DNA bound by polylysine or inversely proportional to the sizes of free DNA loops. A of large amount of renaturation of free base pairs has also been observed for 0.6 M and 1.6 M NaCl-treated nucleohistones. The binding of polylysine to nucleohistone enhances the renaturation of histone-bound base pairs. The percent of renaturation of polylysine–bound base pairs is high and is approximately independent of the extent of binding on DNA by polylysine. This is true in polylysine–DNA complexes prepared either by reconstitution or by directly mixing. It also applies for polylysine–nucleohistone complexes. The model where polylysine-bound base pairs collapse at T_m′ with two complementary strands still bound by polylysine is favored over the model where polylysine is dissociated from DNA during melting. The low renaturation of histone-bound base pairs in nucleo-histone indicates that either histones do not hold two complementary strands of DNA tightly or that histones are fully or partially dissociated from DNA when the nucleo-histone is fully denatured.     

Heterogeneity of mitochondrial DNA from Saccharomyces carlsbergensis: renaturation and sedimentation studies

The renaturation kinetics of mitochondrial DNA from the yeast Saccharomyces carlsbergensis have been studied at different temperatures and molecular weights. At renaturation temperatures 25 deg. C below the mean denaturation temperature (T_m) in 1 M-sodium chloride the renaturation rate constant is found to decrease with increasing molecular weight of the reacting strands. This unusual molecular weight dependency gradually disappears with an increase in the renaturation temperature. At a temperature 10 deg. C below the melting point, the rate constant shows the normally expected increase with the square root of the molecular weight. From the renaturation data at this temperature, the molecular weight of the mitochondrial genome is estimated to be about 5·0 × 10⁷. The same size of genome was found from renaturation at low molecular weight and 25 deg. C below the T_m.The sedimentation properties of denatured mitochondrial DNA at pH values 7·0 to 12·5 were used to study the conformation of this DNA in 1 M-sodium chloride. The results obtained support the conclusion from the renaturation studies: that the pieces of denatured mitochondrial DNA with a molecular weight above 2 × 10⁵ to 3 × 10⁵, in 1 M-sodium chloride at 25 deg. C below the mean denaturation temperature are not fully extended random coils. Presumably, interaction between adenine and thymine-rich sequences, which are clustered at certain distances within the molecules, is the molecular basis for these observations.     

Further analysis of the altered secondary structure of superhelical DNA. Sensitivity to methylmercuric hydroxide a chemical probe for unpaired bases

A previous study in our laboratory of the reaction of formaldehyde with super-helical DNAs (φX replicative form and PM2) has led to a model for superhelical DNA in which there is a region or regions of altered secondary structure containing unpaired bases. Similar experiments using the nicked circular DNA gave no evidence of interruptions of base pairing. In this study we present additional data, which support the above model as well as extending our analysis of the secondary structure of superhelical DNA and the dynamics of the early denaturation process. In a series of experiments involving the binding of methyl-mercury as a chemical probe of unpaired bases, we obtained the following results. (1) Initially, both s⁰_20w and the buoyant density of the superhelical form of phage PM2 DNA increased as a function of methylmercuric hydroxide concentration, whereas the nicked form did not. (2) This initial binding is accompanied by an increase in superhelical content τ from ?41 to ?46 turns. (3) The binding analysis allows us to estimate that 3.7% of the bases contain methylmercury in this phase of the transition. This is in excellent agreement with the extent of formylation. (4) Such a preformylated molecule shows a shift in the transition to lower mercurial concentrations. These results are interpreted as follows. The initial increase in ?τ excludes the possibility that binding occurs to normal base-paired structures, since this would produce a coupled unwinding of duplex and superhelical turns. The additive effects of formylation and methylmercury binding support the concept that both chemical probes attack the same sites and induce similar structural changes. Thus the evidence clearly supports the view that superhelical DNA contains localized region(s) of interrupted base pairing. Recent studies from other laboratories using single strand-specific endonucleases are in complete agreement with this model.     

Hysteresis and partial irreversibility of denaturation of DNA as a means of investigating the topology of base distribution constraints: application to a yeast rho- (petite) mitochondrial DNA

Low salt concentrations prevent reassociation of separated single strands of DNA, but not the renaturation of partially melted molecules. Rewinding, however, may be delayed (hysteresis) and/or incomplete (partial irreversibility). Long-range fluctuations in base compositioncould account for these observations: (a) the “zippering-up” of a denatured (G + C)-rich section may have to await that of one of its neighbouring (A + T)-rich sections, hence a temperature lag in rewinding; (b) the removal of intramolecular heterogeneities in base composition by fragmentation will give rise to a dispersal of strand-separation temperatures. Conversely, it is shown how a considerable amount of information about the topology of base distribution constraints could be derived from these phenomena.Some yeast ρ^? (petite) mitochondrial DNAs, the melting of which is quasidiscontinuous, provide an excellent opportunity for testing the applicability of this new approach to denaturation mapping. Alternating partial denaturation and renaturation with a low rate of temperature change were followed by high-frequency recording of absorbance at 260 nm. A typical experiment (counterion concentration 0.015 m-Na⁺) carried out on a low-complexity (length of repetitive unit about 3000 base-pairs) ρ^? DNA is reported in full detail. Analysis of the data disclosed the existence of two relatively (G + C)-rich clusters separated by long homogeneous stretches of high (A + T) content.The rewinding of ρ^? DNAs is a discontinuous process. Both equilibrium and non-equilibrium melting processes were observed. Hysteresis in rewinding, which is restricted to the melting range, increases discontinuously with the extent of unwinding reached prior to cooling. Results are shown to be fully consistent with a model that presupposes that nucleation does not play any part in the renaturation process. They are briefly discussed further in the light of current concepts in the theory of helix-coil transitions of DNA.     

Thermal Denaturation of β-Glucosidase B from <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> Proceeds Through a Lumry-Eyring Mechanism

β-glucosidase B (BglB), 1,4-β-d-glucanohydrolase, is an enzyme with various technological applications for which some thermostable mutants have been obtained. Because BglB denatures irreversibly with heating, the stabilities of these mutants are assessed kinetically. It, therefore, becomes relevant to determine whether the measured rate constants reflect one or several elementary kinetic steps. We have analyzed the kinetics of heat denaturation of BglB from Paenibacillus polymyxa under various conditions by following the loss of secondary structure and enzymatic activity. The denaturation is accompanied by aggregation and an initial reversible step at low temperatures. At T ≥ T _m , the process follows a two-state irreversible mechanism for which the kinetics does not depend on the enzyme concentration. This behavior can be explained by a Lumry-Eyring model in which the difference between the rates of the irreversible and the renaturation steps increases with temperature. Accordingly, at high scan rates (≥1 °C min⁻¹) or temperatures (T ≥ T _m ), the measurable activation energy involves only the elementary step of denaturation.     

The kinetics of the renaturation of deoxyribonucleic acid denatured in the presence of copper(II) ions

DNA which has been heat denatured in the presence of Cu⁺⁺ ions can be completely and rapidly renatured by increasing the ionic strength of the solution above a critical value. A kinetic study of this renaturation recation was carried out by following the associated UV absorbance change and also by following the change in free Cu⁺⁺ ion concentration by means of a specific Cu⁺⁺ ion activity electrode. The data obtained could be fitted to first-order kinetics for a considerable extent of the reaction and the rate constant was found to increase with temperature and ionic strength, but to decrease markedly as the bulk viscosity of the solution was increased. At temperatures greater than 5°C the reaction rate depended on the time elapsing between denaturation and the commencement of the renaturation reaction. As there was good agreement between the rate constants obtained by following the decrease in hyperchromism and by following the increase in free Cu⁺⁺ ion concentration, it is concluded that under the conditions employed, the rate of renaturation is determined by the rate of release of Cu⁺⁺ ions from the denatured DNA-Cu⁺⁺ complex.     

Thermodynamics and kinetics of the interaction of copper (II) ions with native DNA.

Based on equilibrium binding studies, as well as on kinetic investigations, two types of interactions of Cu²⁺ ions with native DNA at low ionic strength could be characterized, namely, a nondenaturing and a denaturing complex formation. During a fast nondenaturing complex formation at low relative ligand concentrations and at low temperatures, different binding sites at the DNA bases become occupied by the metal ions. This type of interaction includes chelate formation of Cu²⁺ ions with atoms N₍₇₎ of purine bases and the oxygens of the corresponding phosphate groups, chelation between atoms N₍₇₎ and O of C₍₆₎ of the guanine bases, as well as the formation of specific intestrand crosslink complexes at adjacent G°C pairs of the sequence dGpC. CD spectra of the resulting nondenatured complex (DNA–Cu²⁺)_nat may be interpreted in terms of a conformational change of DNA from the B-form to a C-like form on ligand binding. A slow cooperative denaturing complex formation occurs at increased copper concentrations and/or at increased temperatures. The uv absorption and CD spectra of the resulting complex, (DNA–Cu²⁺)_denat, indicate DNA denaturation during this type of interaction. Such a conclusion is confirmed by microcalorimetric measurements, which show that the reaction consumes nearly the same amount of heat as acid denaturation of DNA. From these and the kinetic results, the following mechanism for the denaturing action of the ligands is suggested: binding of Cu²⁺ ions to atoms N₍₃₎ of the cytosine bases takes place when the cytosines come to the outside of the double helix as a result of statistical fluctuations. After the completion of the binding process, the bases cannot return to their initial positions, and thus local denaturation at the G·C pairs is brought about. The probability of the necessary fluctuations occurring is increased by chelation of Cu²⁺ ions between atoms N₍₇₎ and O of C₍₆₎ of the guanine bases during nondenaturing complex formation, which loosens one of the hydrogen bonds within the G·C pairs, as well as by raising the temperature. The implications of the new binding model, which comprises both the sequence-specific interstand crosslinks and the described mechanism of denaturing complex formation, are discussed and some predictions are made. The model is also used to explain the different renaturation properties of the denatured complexes of Cu²⁺, Cd²⁺, and Zn²⁺ ions with DNA. In temperature-jump experiments with the nondenatured complex (DNA–Cu²⁺)_nat, a specific kinetic effect is observed, namely, the appearance of a lag in the response to the perturbation. The resulting sigmoidal shape of the kinetic curves is considered to be a consequence of the necessity of disrupting a certain number of the crosslinks existing in the nondenatured complex before the local unwinding of the binding regions (a main step of denaturing complex formation) may proceed.     

Excluded volume effects on the rate of renaturation of DNA

The rate of renaturation of T2 DNA hits been investigated by using complementary DNA strands of different length. The length of the shorter strand ranged from 0.02 to 1.0 times the length of the longer strand. An excluded volume theory is developed to include this type of reaction as well as the DNA–RNA hybridization reaction. Experimental and theoretical rates of renaturation of DNA are found to be in agreement. For the cases studied, the rate was never greater than twice that observed for short strands of the same length renaturing with themselves. The products of renaturation reactions are also considered.     

Transfection of Escherichia coli by Mu DNA

Summary Infectivity of Mu DNA was demonstrated in Ca⁺⁺-treated Escherichia coli cells that lacked the nucleases Exo V and Endo I. The efficiency of transfection is about 10^-7 per phage equivalent. Infectivity is destroyed by denaturation of Mu DNA, and cannot be restored by renaturation.     

Chromosomal DNA denaturation and reassociation in situ.

The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10^?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization.     

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.     

The Kinetics of DNA Helix-Coil Subtransitions

Abstract

The kinetic analysis of individual helix-coil subtransitions was performed by comparing melting and renaturation profiles obtained at different temperature change rates. The duration of the three transition stages and its dependence on temperature and ionic strength were determined for a T7 phage DNA fragment. The obtained temperature dependence of the melting time for a stretch flanked by melted regions is in quantitative agreement with that predicted by the theory of slow processes (V.V. Anshelevich, A.V. Vologodskii, A.V. Lukashin, M.D. Frank-Kamenetskii, Biopolymers 23, 39 (1984)). The reasons are discussed for the increasing relaxation time of this stretch in the middle of its transition with decreasing ionic strength.

The zipping kinetics of a melted region under essentially nonequilibrium conditions was examined for T7 fragment and pAO3 DNAs. The obtained temperature dependence of the zipping time is in quantitative agreement with calculations based on the theory of slow processes.

The renaturation times of stretches flanked by helical regions proved fairly small even at a low ionic strength. These times are several orders of magnitude smaller than the renaturation times of the same stretches with one helical boundary. A formal application of the theory of slow processes failed to account for the small renaturation times of stretches that are zipped from both ends. This is probably due to the non-allowance for the changing entropy of the loop linking two helix-coil boundaries migrating towards each other.

Slow processes have been revealed in the intramolecular melting of Col E1 DNA at a high ionic strength. The reason for the long relaxation time of one subtransition is the large size of the loop that separates the melting stretch from the helical part of the molecule. This result can be accounted for by the theory of slow processes.     

A Mesoscale Model of DNA and Its Renaturation

A mesoscale model of DNA is presented (3SPN.1), extending the scheme previously developed by our group. Each nucleotide is mapped onto three interaction sites. Solvent is accounted for implicitly through a medium-effective dielectric constant and electrostatic interactions are treated at the level of Debye-Hückel theory. The force field includes a weak, solvent-induced attraction, which helps mediate the renaturation of DNA. Model parameterization is accomplished through replica exchange molecular dynamics simulations of short oligonucleotide sequences over a range of composition and chain length. The model describes the melting temperature of DNA as a function of composition as well as ionic strength, and is consistent with heat capacity profiles from experiments. The dependence of persistence length on ionic strength is also captured by the force field. The proposed model is used to examine the renaturation of DNA. It is found that a typical renaturation event occurs through a nucleation step, whereby an interplay between repulsive electrostatic interactions and colloidal-like attractions allows the system to undergo a series of rearrangements before complete molecular reassociation occurs.     

DNA melting temperatures and renaturation rates in concentrated alkylammonium salt solutions

DNA melting temperatures and renaturation rates have been determined for Me₂Et₂NBr and a series of RMe₃NBr and REt₃NBr solvents where R is a linear hydrocarbon chain. The point of independence of DNA melting temperature on base composition has been investigated for each solvent system. Renaturation rates are compared with those found in other concentrated salt solutions. Solvent mixtures which accelerate DNA renaturation have also been investigated.     

Kinetics of renaturation of denatured DNA. II. Products of the reaction

The structure of renatured T4 DNA has been studied by CsCl density-gradient centrifugation. It has been found that the products of the reaction differ, depending on the method used for denaturation of the DNA. If denaturation is carried out without taking precautions to prevent chain degradation, for example, by heat, the DNA formed by renaturation shows approximately 70% recovery of the native structure as judged by its density. With long times of annealing, the DNA can recover the native density. This behavior is also observed with bacterial DNA samples. On the other hand, if precautions arc taken to prevent chain degradation during denaturation, two products appear as a result of renaturation. One of them is undistinguishable from native T4 DNA, whereas the second one consists of highly aggregated DNA which shows only a partial recovery of the native structure. With long times of annealing, this second species recovers the native density but retains its highly aggregated nature. At higher ionic-strengths, renaturation follows a different pattern and a single product is formed. The relevance of all these observations to the kinetic anomalies reported in the previous communication is discussed.     

Renaturation of DNA in the presence of ethidium bromide

The rate of renaturation of T2 DNA has been studied as a fuction of ethidium bound per nucleotide of denatured DNA. The Binding constants and number of binding sites for ethidium have been determined by spectral titration for denatured DNA at 55, 65, and 75°C and for native DNA at 65°C in 0.4M Na⁺. The rate of renaturation of T2 DNA was found to be independentof ethidium binding up to 0.03 moles per mole of nucleotide. Above 0.03 moles, the rate drops off precipitously approaching zero at 0.08 and 0.06 moles bound ethidium per nucleotide at 65°C respectively. A study was also made of the use of bound ethidium fluorescence as a probe for monitoring DNA renaturation reactions.     

Variation in satellite DNA from some higher plants

Pure satellite DNAs were prepared as minor components after centrifugation to equilibrium on CsCl gradients. A single satellite DNA band was isolated from flax (Linum usitatissimum) DNA and two bands were resolved in cucumber (Cucumis sativus) DNA. These apparently homogeneous components of the plant genomes were further analyzed by thermal denaturation and renaturation. The flax satellite DNA appeared homogeneous on thermal denaturation but was shown to contain several components of renaturation. The two cucumber satellite DNAs were different from each other, but both showed at least two components in denaturation and renaturation analyses. Renaturation in the three satellites, particularly in flax, was inaccurate, indicating a considerable degree of sequence divergence. Although each satellite contained quite large amounts of simple repetitious sequences, a residual heterogeneous DNA fraction was always present. It is considered that this was too large a portion of the satellite DNA to be due to organelle or ribosomal DNA in cucumber. The latter possibility is precluded in flax, where the satellite is completely resolved in buoyant density from both organelle and ribosomal DNA.