Photodynamic activity of dyes with different DNA binding properties II. T4 phage inactivation.

The photosensitizing efficiency of six dyes--proflavine, 9-aminoacridine, ethidium bromide, thiopyronine, pyronine and acridine red--have been compared on the basis of the inactivation of sensitized T4 phage caused by light irradiation. This reaction was only measurable after diffusion of the dye through the phage capsid and was not observed in the presence of either chloroquine or quinacrine; it followed a single-hit kinetics as a function of the irradiation time. With each dye, a double reciprocal plot of the inactivation constant versus the dye concentration present gave rise to a linear relationship. From this relation, parameters were deduced which expressed the relative photosensitizing efficiencies. Dye-binding to the phages was measured and the proflavine-mediated inactivation appeared to be related to the amount of strongly bound molecules. Such a conclusion could not be reached in the case of 9-aminoacridine and ethidium bromide, which were much less efficient photosensitizers than proflavine, but which were also strongly bound to the phages. Thiopyronine was weakly bound to the phages; it had, however, the highest photosensitizing activity observed. These results indicate that various mechanisms are involved when the phage photosensitization is due to one dye or another.     

Induced circular dichroism of DNA-dye complexes

The binding of methylene blue, proflavine, and ethidium bromide with DNA has been studied by spectrophotometric titration. Methylene blue and proflavine or methylene blue and ethidium bromide were simultaneously titrated by DNA. The results indicate that all of these dyes compete for the same bindine sites. The binding properties are discussed in terms of symmetry. The optical properties of the dye–DNA complexes have been studied as a function of DNA/dye ratio. The induced circular dichriosm due to dye–dye interaction was measured at low dye/DNA ratios for cases involving both the same dye and different dyes. A positive Cotton effect for DNA–proflavine complex may be induced at 465 mμ by eithr proflavine or ethidium bromide, whereas a netgative Cotton effect at 465 mμ may be induced by methylene blue. The limiting circular dichroism, with no dye–dye interaction, and the induced circular dichroism spectra are discussed in terms of symmetry rules.     

Calorimetry of DNA-dye interactions in aqueous solution: I. Proflavine and ethidium bromide

The results of an investigation on the interaction of proflavine and of ethidium bromide with DNA (calf thymus) in dilute aqueous solution are reported. The binding of the two dyes by DNA has been studied by means of microcalorimetric and of equilibrium dialysis measurements. Data on the thermodynamics of dimerization of both proflavine and ethidium bromide in aqueous solution obtained on the basis of spectroscopic and/or calorimetric experiments are also reported.The enthalpy data show that dye-dimerization and dye “strong” interaction with DNA are energetically favourable and quite similar while only in the latter case the phenomenon is also entropy driven. This is taken as further evidence in support of the concept that “strong” interaction-of both proflavine and ethidium bromide with DNA means dye molecules intercalation into the native, double helical structure of the biopolymer.     

Quantitative analysis of fluorescence profiles of chromosomes : Influence of DNA base composition on banding

Chromosome banding has been analysed in terms of DNA content and base composition distribution along five human chromosomes. Three intercalative dyes (quinacrine, proflavine and ethidium bromide) whose fluorescence quantum yield in the presence of DNAs of different base compositions has been determined, have been used to examine the influence of base composition on the chromosome patterns. Considering that the amount of DNA as determined by the Feulgen reaction is almost constant along the chromosome arms and assuming that base composition is the only factor influencing the fluorescence of these dyes, a distribution of the A-T base pair content along the chromosomes has been calculated from the fluorescence intensity profiles. From the ratio of the intensity profiles obtained with quinacrine and proflavine, patterns showing the variation of the DNA content and of the A-T base pair content could also be obtained independently. The validity of these different approaches is discussed.     

Internal motion of DNA in bacteriophages

We have investigated internal motion of DNA in bacteriophages by measuring fluorescence anisotropy decays of intercalated ethidium. The results showed large suppression of the internal motion of the inner DNA; the interhelix interaction of the DNA in the phage head is considered to enhance the effective viscosity of the DNA rod and to restrict the angle of the internal motion. Considering that the observed internal motion arises mainly from torsional motion of the DNA, we have calculated the movable angles of the torsional motion (the standard deviation of the torsional motion) of the DNA in the phage heads. The magnitude of the calculated movable angles indicates the extent of suppression of the DNA movement in the phage head; in lambda wild type phage, the DNA is packed most rigidly in the head and the motion is found to be restricted most severely. In a deletion mutant of lambda phage, whose inner DNA content is deficient by 17.6%, steric hindrance from the interhelix DNA interaction is decreased, and the DNA can move more easily. In T4 wild type phage, although the extent of condensation of the inner DNA is the same as that in lambda wild type phage, the DNA was fairly mobile. The presence of glucosylated hydroxymethylcytosine is suggested to influence the rigidity of the inner DNA or packaging mode of the DNA in the T4 head.     

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.     

Intracellular events during infection by Haemophilus influenzae phage and transfection by its DNA

Intracellular events following infection of competent Haemophilus influenzae by HPlcl phage, or transfection by DNA from the phage, were examined. Physical separation of a large fraction of the intracellular phage DNA from the bulk of the host DNA was achieved by lysis of infected or transfected cells with digitonin, followed by low-speed centrifugation. The small amount of bacterial DNA remaining with the phage DNA in the supernatants could be distinguished from phage DNA by its ability to yield transformants. After infection by whole phage, three forms of intracellular phage DNA were observable by sedimentation velocity analysis: form III, the slowest-sedimenting one; form II, which sedimented 1.1 times faster than III, and form I, which sedimented 1.6 times faster than III. It was shown by electron microscopy, velocity sedimentation in alkali, and equilibrium sedimentation with ethidium bromide, that forms I, II and III are twisted circles, open circles, and linear duplexes, respectively.After the entry of phage DNA into wild-type cells in transfection, the DNA is degraded at early times, but later some of the fragments are reassembled, resulting in molecules that sediment faster than the monomer length of phage DNA. Some of the fast-sedimenting molecules are presumably concatemers and are generated by recombination. In strain rec₁^? the fast-sedimenting molecules do not appear and degradation of phage DNA is even more pronounced than in wild-type cells. In strain rec₂^? there is little degradation of phage DNA, and the proportion of fast-sedimenting molecules is much smaller than in wild-type cells. Since rec₁^? and rec₂^? are transfected with much lower efficiency than wild type, our hypothesis is that both fragmentation and generation of fast-sedimenting phage DNA by recombination are required for more efficient transfection.     

Direct transfer of coliphage lambda DNA from Escherichia coli to cellular slime mold Dictyostelium discoideum

Dictyostelium discoideum myxamoebae were cultured with Escherichia coli cells infected with lambda phage in the presence of chloramphenicol. After eliminating the uningested bacteria by repeated centrifugation in a Percoll gradient, we examined the myxamoeba cytoplasm (not the food vacuole) for the presence of phage DNA. A significant amount of DNA extracted from the myxamoebae was hybridizable with purified phage lambda DNA, and capable of forming phage particles when packaged in vitro with phage lambda proteins. The EcoRI restriction maps of the phages recovered from the plaques were identical to that of the infecting phage. These results strongly suggest that phage DNA molecules were taken up by the cellular slime mold cells and that at least some fraction existed in intact form.     

Effect of organic solvents on the properties of the complexes of DNA with proflavine and similar compounds

In order to obtain information on the binding forces involved in the formation of the complex proflavine–DNA by the stronger process I, the stability of the complexes was investigated in the presence of various organic solvents, methanol, ethanol, n-propanol, isopropanol, formamide, dimethyl sulfoxide, p-dioxane, glycerol, and ethylene glycol. Quantitative data on binding in terms of K/n and r were obtained by means of absorption and fluorescence spectra, as well as by a thermal denaturation technique. All organic solvents used decrease the binding ability of the dye. The effectiveness of the solvents increases with their hydrocarbon content, but can hardly be related to their dielectric constant. The complex formation is effectively suppressed by organic solvent concentrations, in which DNA still preserves its double-helical conformation. These results demonstrate the importance of hydrophobic forces in the formation of the complex proflavine–DNA in aqueous solution. The similarity in spectroscopic properties of proflavine bound to DNA by process I and the same dye dissolved in an organic solvent make it possible to interpret the observed red shift of the long-wavelength absorption peak as being due to the interaction of the dye molecules with the less polar environment. The same behavior was found for other dyes capable of intercalation like purified trypaflavine, phenosafranine and ethidium bromide. However, intercalation is not a necessary condition, as it was shown in the case of pinacyanol, which binds only at the surface of DNA.     

Investigation of DNA dynamics and drug-DNA interaction by steady state fluorescence anisotropy.

We have used steady-state fluorescence polarization anisotropy (FPA) of ethidium probe molecules bound to DNA to investigate DNA-DNA interactions and the effect of high densities of intercalating drugs on the internal motions of DNA responsible for depolarization of the ethidium fluorescence. To calibrate the method, we examined the effect of DNA length on (FPA) using DNA varying in size from 10-150 base pair. The association of approximately 30 base pair DNA at high concentrations was then detected by its effect on (FPA). With sample concentrations approaching those commonly used in various physical experiments (NMR, Raman) significant DNA-DNA interactions are observed. With high molecular weight DNA (greater than 500 base pair), the limiting value of the (FPA) (0.23) is due to internal motions of the DNA (and bound chromophores). The (FPA) of ethidium probe molecules (1 drug/200 base pair) is unaffected by the addition of high levels (1 drug/2 base pair) proflavine. This indicates that either the elastic properties of DNA are unaffected by high densities of intercalated drug or that the depolarization of the ethidium fluorescence is due to highly localized motions of the base pairs that are unperturbed by binding of drugs at neighboring sites.     

Luria effect in bacteriophages and the role of the products of recA+ and lexA+ genes in its induction

An analysis of UV-damages accumulation in the phages as revealed by delay of intracellular growth is represented using temperate lambda phage. The maximum of growth delay of phage lambda at given UV-dose was found with lambda red+, infecting Escherichia coli AB1886 uvrA strain. The growth delay was absent, when a strain RH-1 uvrA-recA- was infected with UV-irradiated phage lambda red3. A moderate growth delay was obtained with the phages lambda red+, infecting E. coli RH-1 uvrA-recA- or phage lambda red3, infecting E. coli AB1886 uvrA-. THe growth delay was also absent when wild type, recA- and uvrA mutants of E. coli were infected with phage lambda after 8-metnoxypsoralen + light (lambda > 310 nm) treatment. It is known that the crosslinks appear to be the DNA defects which give rise to the observed biological inactivation following psoralen + light treatment. However, a considerable growth delay of phage lambda, treated by 8-metnoxypsoralen + light, was only found under condition of crosslinks repair (W-reactivation and prophage-reactivation). The results obtained are best explained by the assumption that the growth delay reflects the time required for the postreplication repair (RecA, LexA, Red) of any lethal UV-lesion.     

Effects of Polyamines on the Binding of Hoechst 33258 to Calf Thymus DNA

Abstract

The ability of polyamines to displace the minor groove-binding dye Hoechst 33258 from calf thymus DNA was investigated. Polyamines displace non-specific DNA phosphate bound Hoechst in a charge-dependent fashion, but show very little ability to displace the high affinity binding of Hoechst in the minor groove of DNA. This high affinity binding is, however, sensitive to ethidium bromide and the minor groove binding drug berenil. These studies suggest that polyamines probably bind DNA in the minor groove very weakly, if at all, relative to known minor groove binding agents.     

Putrescine and certain polyamines can inhibit DNA injection from bacteriophage lambda

The injection of λDNA from attached phage into a host bacterium can be reversibly inhibited by putrescine. The concentration of di- or polyamine required to inhibit injection varies with the Mg²⁺ concentration and the amount of DNA in the phage head. In a series of n-alkyl diamines, those with more than five or fewer than three CH₂ groups between amino groups were ineffective.     

Increased DNA polymerase and ATP-dependent deoxyribonuclease activities following DNA damage in Mycobacterium smegmatis

Summary Treatment of growing cultures of Mycobacterium smegmatis with alkylating agents (methyl methaneusulphonate, ethyl methanesulphonate, nitrogen mustard, or mitomycin C) or with ultraviolet light resulted in enhanced specific activities of a DNA polymerase and of an ATP-dependent deoxyribonuclease. Similar results had previously been obtained with hydroxyurea and with iron limitation. The three of these treatments which were tested (methyl methane-sulphonate, mitomycin C and hydroxyurea) produced strand breaks or alkali-labile regions in the DNA of this organism. The increased enzyme activities could be prevented by simultaneous treatment with inhibitors of protein synthesis.In contrast, treatment of the cultures with intercalating agents (ethidium bromide, acridine orange, or proflavine), 5-fluorouracil, caffeine, or nalidixic acid, inhibited DNA synthesis without increasing the enzyme activities. These treatments did not produce strand breaks in the DNA of this organism.The results support the hypothesis that, in M. smegmatis, damage to DNA induces increased synthesis of enzymes associated with DNA repair.     

Canavanine-mediated depletion of polyamine pools in Escherichia coli: effect of head morphogenesis and DNA synthesis.

We have found that L-canavanine inhibited the synthesis of polyamines in T4-infected Escherichia coli. These polyamines are known to be required for T4 DNA synthesis and may be involved in phage morphogenesis. The new data indicate that the inhibition of polyamine synthesis is not primarily responsible for the L-conavanine-mediated inhibition of DNA synthesis nor does it seem to be involved in the induction of lollipops. L-Canavanine does influence the relative amounts of putrescine and spermidine found in the phage particle, but it does not influence the amount of DNA phosphate neutralized by polyamines.     

Analysis of the coliphage T5 DNA ejection process with free and liposome-associated TonA protein.

Outer membrane protein TonA, the receptor for coliphage T5, has been partially purified and incorporated into the phospholipid bilayer of liposomes. Adsorption of the phage to its receptor in either a free or liposome-associated form is fast and sufficient to trigger the ejection of encapsidated DNA. In both in vitro systems the exit of DNA from the phage capsid is a very slow process. Ejected DNA can partially accumulate inside the liposome aqueous compartment, but the transfer from the phage head to the liposome internal space is never complete, perhaps because the liposome volume is too small. The presence of polyamines or divalent cations (magnesium) or both in the incubation medium diminished the extent of DNA ejection, possibly by stabilizing DNA inside the head. DNA movement was slowed as the temperature was decreased from 37 to 18 degrees C. Furthermore, incubation at 4 degrees C totally prevented this DNA movement, even if a large part of the DNA had already exited the capsid.     

Mechanism of inhibition of bacteriophage T7 DNA synthesis in Escherichia coli B cells infected by alkylated bacteriophage T7

Quantitative analysis of DNA replication, in E. coli B cells infected by methyl methanesulfonate-treated bacteriophage T7, showed that production of phage DNA was delayed and decreased. The cause of the delay appeared to be a delay in host-DNA breakdown, the process which provides nucleotides for phage-DNA synthesis. In addition, reutilisation of host-derived nucleotides was impaired. These observations can be accounted for by a model in which methyl groups on phage DNA slow down DNA injection and also reduce the replicational template activity of the DNA once it has entered the cell. Repair of alkylated phage DNA may be required not only for replication but also for normal injection of DNA.     

Comparative studies of the effects of acridines and other petite inducing drugs on the mitochondrial genome of Saccharomyces cerevisiae.

Summary The effects of the acridines euflavine and proflavine on mitochondrial DNA (mtDNA) replication and mutation inSaccharomyces cerevisiae have been compared. In contrast to previous results we found that under our conditions proflavine can indeed induce high levels (>80%) of petite mutants, although six times less efficiently than euflavine. The parameters measured for mutagenesis of the mitochondrial genome and inhibition of mtDNA replication in whole cells suggest that the modes of action of euflavine and proflavine are very similar. After extended (18h) treatment of growing cells with each drug the percentage loss of mtDNA or genetic loci was almost coincidental with the extent of petite induction.It was found that proflavine is equally as effective as euflavine in inhibiting mtDNA replication in isolated mitochondria in contrast to the differential between the drugs observed in vivo. However, proflavine and euflavine inhibit cellular growth at almost the same concentrations. It is therefore proposed that there is some intracellular permeability barrier which impedes proflavine access to the mitochondrial DNA replicating system.The petites induced by euflavine (and proflavine) are characterized by there being a preferential induction ofrho ⁰ petites lacking mtDNA as opposed torho ^- petites retaining mtDNA. This is in contrast to the relative proportions of such petites induced by ethidium bromide or berenil. A scheme for the production of petites by euflavine is presented, in which euflavine inhibits the replication of mtDNA, but does not cause direct fragmentation of mtDNA (unlike ethidium bromide and berenil). The proposed scheme explains the production of the high frequency ofrho ^o cells, as well as therho ^- cells induced by euflavine. The scheme also accounts for previous observations that euflavine only mutants growing cultures, and that the buds, but not mother cells, become petite.     

Folding of prokaryotic DNA. Isolation and characterization of nucleoids from Bacillus licheniformis

Intact and fast-sedimenting nucleoids of Bacillus licheniformis were isolated under low-salt conditions and without addition of detergents, polyamines or Mg2+. These nucleoids were partially unfolded by treatment with RNase and completely unfolded by treatments that disrupt protein-DNA interactions, like incubation with proteinase K, 0.1% sodium dodecyl sulphate and high ionic strength. Ethidium bromide intercalation studies on RNase-treated, proteinase-K-treated and non-treated nucleoids in combination with sedimentation analysis of DNase-I-treated nucleoids revealed that DNA is organized in independent, negatively supertwisted domains. In contrast to the DNA organization in bacterial nucleoids, isolated under high-salt conditions and in the presence of detergents (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972), the domains of supertwisted DNA in the low-salt-isolated nucleoids studied here are restrained by protein-DNA interactions. A major role for nascent RNA in restraining supertwisted DNA was not observed. The superhelix density of B. licheniformis nucleoids calculated from the change of the sedimentation coefficient upon ethidium bromide intercalation, was of the same order of magnitude as that of other bacterial nucleoids and eukaryotic chromosomes, isolated under high-salt conditions: namely, -0.150 (corrected to standard conditions: 0.2 M-NaCl, 37 degrees C; Bauer, 1978). Electron microscopy of spread nucleoids showed relaxed DNA and regions of condensed DNA. Spreading in the presence of 100 micrograms ethidium bromide per ml revealed only condensed structures, indicating that nucleoids are intact. From spreadings of proteinase-K-treated nucleoids we infer that supertwisted DNA and the protein-DNA interactions, responsible for restraining the superhelical DNA conformation, are localized in the regions of condensed DNA.     

The intracellular growth of bacteriophages. I. Liberation of intracellular bacteriophage T4 by premature lysis with another phage or with cyanide

A method is described for liberating and estimating intracellular bacteriophage at any stage during the latent period by arresting phage growth and inducing premature lysis of the infected cells. This is brought about by placing the infected bacteria into the growth medium supplemented with 0.01 M cyanide and with a high titer T6 lysate. It was found in some of the later experiments that the T6 lysate is essential only during the first half of the latent period. Cyanide alone will induce lysis during the latter part of the latent period. Using this method on T4-infected bacteria it is found that during the first half of the latent period no phage particles, not even those originally infecting the bacteria, are recovered. This result is in agreement with the gradually emerging concept that a profound alteration of the infecting phage particle takes place before reproduction ensues. During the second half of the latent period mature phage is found to accumulate within the bacteria at a rate which is parallel to the approximately linear increase of intracellular DNA in this system. However, the phage production lags several minutes behind DNA production. When 5-methyltryptophan replaced cyanide as the metabolic inhibitor, similar results were obtained. The curves were, however, displaced several minutes to the left on the time axis. The results are compared with Latarjet's (16) data on x-radiation of infected bacteria and with Foster's data (18) concerning the effect of proflavine on infected bacteria. Essential agreement with both is apparent.