Joint molecule formation following conjugation in wild type and mutant Escherichia coli recipients

The sedimentation coefficients of closed circular Simian virus (SV40) DNA, phage PM2 DNA and animal mitochondrial DNAs in alkaline NaCl and alkaline CsCl were found to decrease by about 5% as the initial superhelix densities decreased from 0.0 to ?0.10, corresponding to a decrease in the degree of strand interwinding from 1.0 to 0.9 net turns per ten base pairs. The small dependence of the appropriately normalized sedimentation coefficients on the degree of strand interwinding is taken to indicate that fully titrated and denatured closed circular DNA is highly supercoiled in a positive sense. This supercoiling results from the spontaneous decrease in the number of secondary turns in the no longer ordered pairs of polynucleotide strands.The measured sedimentation coefficients form a smoothly connected monotonie curve when plotted along with the sedimentation coefficients in alkali (Sebring et al., 1971) of parental closed circles derived from closed circular SV40 DNA replicating intermediates. These DNAs have degrees of strand interwinding that range from 0.6 to 0.15.The possibility raised by Paoletti &; LePecq (1971) that closed circular duplex DNAs contain positive supercoils, i.e. have degrees of strand interwinding greater than 1.0, has been ruled out in a series of ethidium bromide titrations of partially replicated mitochondrial DNA before and after removal of the progeny strand. More ethidium bromide was required in the latter case for relaxation, a result which shows that intercalated ethidium and a displacing strand act on the duplex in the same way, and that both unwind the duplex. This result requires the supercoils of naturally closed circular DNAs to be negative.     

Effect of Bending Strain on the Torsion Elastic Constant of DNA

The torsion constants of both circular and linear forms of the same 181 bp DNA were investigated by time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The ratio of intrinsic ethidium binding constants of the circular and linear species was determined from the relative fluorescence intensities of intercalated and non-intercalated dye in each case. Possible changes in secondary structure were also probed by circular dichroism (CD) spectroscopy. Upon circularization, the torsion constant increased by a factor of 1.42, the intrinsic binding constant for ethidium increased by about fourfold, and the CD spectrum underwent a significant change. These effects are attributed to an altered secondary structure induced by the bending strain. Quantitative agreement between torsion constants obtained from the present FPA studies and previous topoisomer distribution measurements on circular DNAs containing 205 to 217 bp removes a long-standing apparent discrepancy between those two methods. After storage at 4°C for eight months, the torsion constant of the circular DNA increased by about 1.25-fold, whereas that of the linear DNA remained unchanged. For these aged circles, both the torsion constant and intrinsic binding constant ratio lie close to the corresponding values obtained previously for a 247 bp DNA by analyzing topoisomer distributions created in the presence of various amounts of ethidium. The available evidence strongly implies that torsion constants measured for small circular DNAs with less than 250 bp are specific to the altered secondary structure(s) therein, and are not applicable to linear and much larger circular DNAs with lower mean bending strains.     

Restricted uptake of ethidium bromide and propidium diiodide by denatured closed circular DNA in buoyant cesium chloride

Alkali-denatured closed circular DNA forms, on neutralization, a relatively stable species first described by Pouwels et al. (1968). In contrast to single-stranded DNA, this denatured two-stranded closed circular DNA species bands densely and co-bands approximately with closed circular duplex DNA in ethidium bromide-CsCl equilibrium density gradients. In CsCl gradients containing propidium diiodide, denDNA I is denser than DNA I, nicked circular DNA and single-stranded φX174 viral DNA. The magnitude of the separations between the above DNAs allows preparative isolation of each when all four are present in the same gradient. The denDNA I has a novel open circular appearance in the electron microscope when cast on standard aqueous hypophases. This species becomes tightly twisted when cast on either aqueous or formamide hypophases containing ethidium bromide. We have concluded from these observations that the high buoyant density of denDNA I in dye-CsCl gradients, relative to single-stranded DNA, is the result of a restricted uptake of dye.     

Ethidium bromide-mediated renaturation of denatured closed circular DNAs. The nature of denaturation-resistant fractions of bacteriophage PM2 closed circular DNA

Addition of the intercalating dye ethidium bromide (EtdBr) to a solution of alkali-denatured double-stranded closed circular PM2, ΦX174, or λb₂b₅c phage DNAs, under conditions such that the solution remains strongly alkaline, can result in the renaturation of up to 100% of the DNA upon neutralization of the solution. For a fixed time of incubation of the alkaline dye-containing solution before neutralization, there exists a minimum concentration of the dye below which no EtdBr-mediated renaturation is observed for each species of closed circular DNA examined. These minimum concentrations increase, for a given DNA, with increasing ionic strength and temperature. The kinetics of accumulation of forms renaturing upon neutralization of alkaline solutions, at fixed concentrations of dye and DNA, are dependent upon the molecular weight and superhelix density of the starting DNA. After extended periods of incubation at a fixed ionic strength and temperature, however, the profiles of percentage of DNA renatured as a function of ethidium concentration become very similar for all the closed circular DNAs tested and display a transition from an absence of dye-mediated renaturation to virtually 100% renaturation upon neutralization over a small range of dye concentration. Circular DNA containing one or more strand scissions remains strand-separated under all the conditions used to effect the renaturation of closed circular DNA. These findings indicate that configurations of closed circular DNA, in which at least some of the complementary bases are apposed, can be selectively stabilized and accumulate in the presence of ethidium in solutions containing 0.19 N hydroxide ion.     

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.     

Ethidium DNA agarose gel electrophoresis: how it started

We started ethidium DNA agarose gel electrophoresis when our ultracentrifuge broke down and we needed an alternative method to check the quality of our mitochondrial DNA preparations. Agarose proved convenient for sizing DNA; ethidium in gel and buffer allowed visualization of DNA bands immediately after the run and improved the separation of the closed and open duplex forms of mitochondrial DNA circles. At smaller gel pore size mitochondrial DNA circles were excluded from the gel, whereas long linear DNAs were not. We concluded that the linear DNAs 'crawl like snakes head on through the gel'. This paper reviews some of the early experiments preceding the introduction of ethidium agarose gel electrophoresis.     

Effect of ethidium on the torsion constants of linear and supercoiled DNAs.

The torsion elastic constants (alpha) of linear pBR322 (4363 bp) and pUC8 (2717 bp) DNAs and supercoiled pBR322 and pJMSII (4375 bp) DNAs are measured in 0.1 M NaCl as a function of added ethidium/base-pair (EB/BP) ratio by studying the fluorescence polarization anisotropy (FPA) of the intercalated ethidium. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single photon counting detection. Previously developed theory for the emission anisotropy is generalized to incorporate rotations of the transition dipole due to excitation transfer. The excitation transfers are simulated by a Monte Carlo procedure (Genest et al., Biophys. Chem. 1 (1974) 266-278) and the consequent rotations of the transition dipole are superposed on the Brownian rotations. After accounting for excitation transfer, the torsion constants of the linear DNAs are found to be essentially independent of intercalated ethidium up to a binding ratio r = 0.10 dye/bp. Dynamic light scattering measurements on linear pUC8 DNA confirm that the torsion constant is independent of binding ratio up to r = 0.20 dye/bp. If alpha d denotes the torsion constant between ethidium and a base-pair, and alpha 0 that between two base-pairs, then our data imply that alpha d/alpha 0 lies in the range 0.65 to 1.64 with a most probable value of 1.0. The torsion constants of supercoiled DNAs decrease substantially with increasing binding ratio even after accounting for excitation transfer. At the binding ratio r* = 0.064, where the superhelix density vanishes and superhelical strain is completely relaxed, the torsion constant of the supercoiled pBR322 DNA/dye complex lies below that of the corresponding linear DNA/dye complex by about 30%. This contradicts the conventional view according to which linear, nicked circular, and supercoiled DNA/dye complexes with r = r* should coexist with the same concentration of free dye, display the same distribution of bound dye, and exhibit identical secondary structures, twisting and bending rigidities, and FPA dynamics. These and other observations imply the existence of metastable secondary structure in freshly relaxed supercoiled DNAs. A tentative explanation is presented for these and other unexpected observations on supercoiled DNAs.     

Densitometric quantification of DNA topoisomers in ethidium bromide-stained agarose gels and chemiluminescence-detected X ray films

This study investigates the contribution of deformational strain imposed by topological interconversions of DNA in ethidium bromide-binding on agarose gels. Closed-circular plasmid DNAs were nicked using UV exposure and the DNA bands were quantified by densitometry. The results show that the closed circular DNA binds the same amount of the dye as its nicked counterpart. The relationship between the band intensity on X-ray films of chemiluminescence-detected Southern blots and DNA concentration was shown to be linear.     

Ethidium bromide-mediated renaturation of denatured closed circular DNAs: mechanistic aspects and fractionation of DNA on a molecular weight basis

When closed circular duplex DNAs are exposed to alkali in the presence of ethidium bromide, from 0 to 100% of the DNA can be recovered as the fully base-paired duplex (native) form upon neutralization of the solutions. The fraction of native DNA depends on the concentration of ethidium bromide, time of incubation, ionic strength and temperature of the solutions before neutralization as well as the molecular weight and superhelix density of the DNA. Limiting ethidium concentrations exist below and above which 0 and 100% of the DNA, respectively, is recovered as native material under a given set of incubation conditions regardless of the length of time of incubation before neutralization. The strong molecular weight dependence of the fraction of DNA recovered in the native form after a given time of pre-neutralization incubation at ethidium concentrations between the limiting values noted above allows larger DNAs to remain fully denatured upon neutralization while smaller DNAs in the same mixture are fully renatured. This permits the rapid fractionation of mixtures of closed duplex DNAs on the basis of molecular weight when a technique for the separation of denatured from fully base-paired DNA is applied to such mixtures. Such a separation has been demonstrated through the marked enrichment of plasmid cloning vector DNA containing cloned inserts in the fractions that remain denatured after neutralization of alkaline solutions of these DNAs containing ethidium bromide.     

Physical properties and gel electrophoresis behavior of R12-derived plasmid DNAs.

A series of closed circular (I) plasmid DNAs has been derived from drug resistance factor R12, and the nicked circular (II) and linear (III) derivatives of these molecules prepared by irradiation in the presence of ethidium bromide and by treatment with restriction enzyme EcoRI, respectively. These DNAs encompass the molecular weight range 3.6 to 61 megadaltons. The base compositions range from 45% to 51% (GC) as estimated by buoyant density determinations. The smaller plasmids are significantly less supercoiled (9-10%) than are the larger (12-13%). The gel electrophoretic behavior of the three DNA structural forms was determined as a function of molecular weight in agarose gels of concentrations ranging from 0.7% to 1.6% and at electrophoresis salt concentrations from 0.02 M to 0.08 M sodium acetate. The mobilities of DNAs I and III undergo a reversal relative to each other at a molecular weight which decreases with increasing agarose gel concentration. The molecular weight at which DNA II fails to enter a gel depends upon the ionic strength during electrophoresis but not upon the gel concentration.     

Molecular recognition of plant DNA: Does it differ from conventional animal DNA?

The recognition mechanism of DNA with small drugs/ligands is an important field of research from pharmacological point of view. Such studies are ample with DNAs extracted from animal cells, but are rare for those extracted from plant cells. However, such a study is strongly demanding for the formulation of pesticides and other agrochemicals. In this contribution, for the first time, we report the interaction of two well-known DNA binder ethidium bromide (EB) and Hoechst 33258 (H33258) with two genomic DNAs extracted from the leaves of Ricinus communis L. (castor bean) and Mangifera indica (mango) using steady-state and picosecond-resolved fluorescence spectroscopy. The purity of the extracted DNAs is confirmed from gel electrophoresis and optical absorption studies. As evidenced from the circular dichroism (CD) measurements the DNAs retain physiologically relevant B forms. The well-known DNA intercalator EB has been found to show an additional electrostatic mode of binding with the DNAs, which is not present in the conventional animal DNAs. The binding affinity of EB is found to be even weaker for the DNA extracted from M. indica compared to that in R. communis L. On the other hand, the binding affinity of H33258 with the plant DNAs is found to be comparable to that of animal DNAs. The difference in interaction could be rationalized from the possible differences in the base sequences.     

Binding of the Biogenic Polyamines to Deoxyribonucleic Acids of Varying Base Composition: Base Specificity and the Associated Energetics of the Interaction

Background

The thermodynamics of the base pair specificity of the binding of the polyamines spermine, spermidine, putrescine, and cadaverine with three genomic DNAs Clostridium perfringens, 27% GC, Escherichia coli, 50% GC and Micrococcus lysodeikticus, 72% GC have been studied using titration calorimetry and the data supplemented with melting studies, ethidium displacement and circular dichroism spectroscopy results.

Methodology/Principal Findings

Isothermal titration calorimetry, differential scanning calorimetry, optical melting studies, ethidium displacement, circular dichroism spectroscopy are the various techniques employed to characterize the interaction of four polyamines, spermine, spermidine, putersine and cadaverine with the DNAs. Polyamines bound stronger with AT rich DNA compared to the GC rich DNA and the binding varied depending on the charge on the polyamine as spermine>spermidine >putrescine>cadaverine. Thermodynamics of the interaction revealed that the binding was entropy driven with small enthalpy contribution. The binding was influenced by salt concentration suggesting the contribution from electrostatic forces to the Gibbs energy of binding to be the dominant contributor. Each system studied exhibited enthalpy-entropy compensation. The negative heat capacity changes suggested a role for hydrophobic interactions which may arise due to the non polar interactions between DNA and polyamines.

Conclusion/Significance

From a thermodynamic analysis, the AT base specificity of polyamines to DNAs has been elucidated for the first time and supplemented by structural studies.     

The inhibition of RNA polymerase by daunomycin

The effect of daunomycin on the in vitro activity of Escherichia coli DNA-dependent RNA polymerase has been studied under a variety of experimental conditions. The inhibition of RNA synthesis by this DNA-binding antibiotic is overcome by an increase in the DNA concentration but is unaffected by an increase in the concentration of the RNA polymerase. It is concluded that, under conditions used, the inhibition is predominantly due to the interaction of the drug with the template DNA. At the concentration used (20 μM), daunomycin is able to inhibit RNA polymerization even after its initiation. However, the possibility remains that other steps are sensitive to daunomycin. A comparison of the effect of daunomycin on RNA synthesis using different DNAs as templates suggests that the extent of inhibition depends on base composition and on the secondary structure of the DNA. The effect of base composition on the melting temperature of antibiotic-DNA complexes is consistent with the inhibiting effect on RNA synthesis.     

Fluorometric methods employing low concentrations of ethidium bromide for DNA topoisomerase and endonuclease assays

DNA topoisomerase activity can be rapidly assayed by measuring the change in ethidium bromide fluorescence intensity after treatment of closed duplex DNA with enzyme. The sensitivity of the fluorometric assay has been enhanced 3-fold by a 10-fold reduction in ethidium bromide concentration to 0.1 microgram/ml. The results of the fluorometric assays are in close agreement with agarose gel electrophoretic analyses of reacted DNA. A sensitive fluorometric method using 0.1 microgram/ml ethidium bromide has also been developed to determine the fraction of nicked and linear DNAs in a mixture containing closed duplex DNA by measuring the fluorescence intensities of ethidium-DNA complexes at pH 7.0 and pH 12.0. These methods make possible very rapid and sensitive measurements of DNA topoisomerase and endonuclease activities.     

Suicide Polymerase Endonuclease Restriction, a Novel Technique for Enhancing PCR Amplification of Minor DNA Templates

PCR-based molecular analyses can be hindered by the presence of unwanted or dominant DNA templates that reduce or eliminate detection of alternate templates. We describe here a reaction in which such templates can be exclusively digested by endonuclease restriction, leaving all other DNAs unmodified. After such a modification, the digested template is no longer available for PCR amplification, while nontarget DNAs remain intact and can be amplified. We demonstrate the application of this method and use denaturing gradient gel electrophoresis to ascertain the removal of target DNA templates and the subsequent enhanced amplification of nondigested DNAs. Specifically, plastid 16S rRNA genes were exclusively digested from environmental DNA extracted from plant roots. In addition, pure culture and environmental DNA extracts were spiked with various amounts of genomic DNA extracted from Streptomyces spp., and selective restriction of the Streptomyces 16S rRNA genes via the suicide polymerase endonuclease restriction PCR method was employed to remove the amended DNA.     

Histone stoichiometry and DNA circularization in archaeal nucleosomes.

Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.