Inhibition of excision repair of DNA in u.v.-irradiated Escherichia coli by phenethyl alcohol

Membrane-specific drugs such as procaine and chlorpromazine have been shown to inhibit excision repair of DNA in u.v.-irradiated E. coli. One possible mechanism is that, if association of DNA with the cell membrane is essential for excision repair, this process may be susceptible to drugs affecting the structure of cell membranes. We examined the effect of phenethyl alcohol, which is a membrane-specific drug and known to dissociate the DNA-membrane complex, on excision repair of DNA in u.v.-irradiated E. coli cells. The cells were irradiated with u.v. light and then held at 30 degrees C in buffer (liquid-holding) in the presence or absence of phenethyl alcohol. It was found that phenethyl alcohol inhibits the liquid-holding recovery in both wild-type and recA strains, corresponding to its dissociating action on the DNA-membrane complex. Thus, the association of DNA with cell membrane is an important factor for excision repair in E. coli. Procaine did not show the dissociating effect, suggesting that at least two different mechanisms are responsible for the involvement of cell membrane in excision repair of DNA in E. coli.     

Process of attachment of phi X174 parental DNA to the host cell membrane

The phi X174-DNA membrane complex was isolated from Escherichia coli infected with phi X174 am3 by isopycnic sucrose gradient centrifugation followed by zone electrophoresis. The phi X174 DNA-membrane complex banded at two positions, intermediate density membrane fraction and cytoplasmic membrane fraction, having bouyant densities of 1.195 and 1.150 g/ml, respectively. Immediately after infection with phi X147, replicating DNA was pulse-labeled and then the incorporated label was chased. The radioactivity initially recovered in the intermediate density membrane fraction migrated to the cytoplasmic membrane fraction. The DNAs from both complexes sedimented mainly at the position of parental replicative form I (RFI). The phi X174 DNA-membrane complex contained a speficic membrane-bound protein having a molecular weigth of 80,000 which is accumulated in the host DNA-membrane complex. These results suggest that when phi X174 DNA penetrated into cells in the early phase of infection, single-stranded circular DNA was converted to parental RFI at a wall/membrane adhesion region and migrated to the cytoplasmic membrane fraction, where the parental RF could serve as a template in the replication of progeny RF.     

Effect of UV radiation on the DNA-membrane complex of Bacillus subtilis]

The influence of UV-light on DNA-membrane complex (DMC) of Bacillus subtilis was studied. An increased DNA content in DMC for strains 168 and rec A-, and a degradation of DMC for strain polA- have been registrated. The increase in DNA in DMC of the two former strains is inhibited by caffeine to be correlated with changes in protein content in DMC, determined by a radioactive label, but not with lipid content. Thus, the association of DNA with the membrane is mediated by proteins. DNA increasing capacity seen in DMC after UV-irradiation and after the following incubation of bacteria in the complete medium is correlated with a relative sensitivity of strains. To explain these data, it is supposed that the reparative synthesis is accomplished in cell on their membranes and that for the normal completion of DNA repair the association between DNA and the membrane is necessary.     

Purification of bacteriophage T7 DNA-membrane complex and its application to the in vitro recombination reaction

In order to construct an in vitro recombination system of T7 DNA, the reaction products of which resemble those in vivo in structure, T7 DNA-membrane complex which is free from concomitant DNase activity was purified from T7 phage-infected cells. T7-infected cells were lysed with T4 lysozyme/Brij58, and T7 DNA-membrane complex was purified through three successive density gradient centrifugations. The properties of the complex on exposure to defined nucleases and observation of the complex by electron microscopy revealed that in T7 DNA-membrane complex, both ends of a linear T7 DNA are bound with membrane components. A mixture of 32P-labeled T7 DNA-membrane complex and BU-labeled T7 DNA-membrane complex was incubated with T7 exonuclease and T7 DNA-binding protein, and the reaction products with intermediate density were purified. Most of the products were found to have structures similar to that of the recombination intermediate found in T7-infected cells upon electron microscopic examination.     

Interactions Between Exogenous Deoxyribonucleic Acid and Membrane Vesicles Isolated from Bacillus subtilis 168

Membrane vesicles isolated from competent cultures of Bacillus subtilis 168 bound up to 20 mug of double-stranded deoxyribonucleic acid (DNA) per mg of membrane protein in the presence of ethylenediaminetetraacetate. The formation of the DNA-membrane complex was time, temperature, and pH dependent. Eighty per cent of the DNA could be removed from the complex by treatment with deoxyribonuclease I. Nevertheless, the DNA that remained attached to the vesicles appeared to have been attacked by the enzyme, suggesting that all the complexed DNA is located at the outer surface of the vesicles. Pretreatment of DNA with deoxyribonuclease I destroyed its affinity for the vesicles. The extent of binding decreased by the addition of Mg(2+) ions, especially at high DNA concentrations (more than 2 mug/ml). This effect was partially due to membrane-associated Mg(2+)-dependent endonucleolytic activity, which caused double-strand breaks in addition to single-strand nicks, and to exonuclease activity. The endonucleolytic activity was enhanced by heating the membranes at 80 C. DNA-membrane association was not markedly affected by sulfhydryl reagents, but was largely inhibited by formaldehyde. Endogenous competence-stimulating activity did not alter the DNA-binding capacity of the vesicles.     

Site of nuclear DNA replication in embryonic cells of the sea urchin Strongylocentrotus intermedius]

In the sea urchin embryonic cells, all newly synthesized nuclear DNA (n-DNA) pulse-labeled by 3H-thymidine was found within DNA-membrane complex (DNA-mc) isolated by centrifugation of lysates of nuclei after their treatment with Sarkosyl, Brij-35, or sodium dodecylsulfate through neutral sucrose (10--30%) gradients. This attachment has been shown not to be an artifact due to the unspecific effect of the detergents or the destabilization of the secondary structure of n-DNA because the association of the exogenous 14C-DNA with nuclear membrane and chromatin did not occur during the isolation of the DNA-mc. n-DNA was not replaced from DNA-mc when the latter was isolated in the excess of unlabeled denatured DNA. n-DNA associated with DNA-mc behaved as a precursor of chromosomal DNA. It is suggested that in sea urchin embryonic cells the synthesis of nuclear DNA is carried out by the replicative complex attached to the nuclear membrane.     

Replication of a low-copy-number plasmid by a plasmid DNA-membrane complex extracted from minicells of Escherichia coli.

A DNA-membrane complex was extracted from minicells of an Escherichia coli mutant harboring a "miniplasmid" derivative (11.2 kilobases) of the low-copynumber plasmid RK2 (56 kilobases). The complex contained various species of supercoiled and intermediate forms of plasmid DNA, of which approximately 20% was bound firmly to the membrane after centrifugation in a CsCl density gradient. The plasmid DNA-membrane complex synthesized new plasmid DNA without the addition of exogenous template, enzymes, or other proteins. DNA synthesis appeared to proceed semi-conservatively, was dependent on the four deoxynucleoside triphosphates, partially dependent on ribonucleoside triphosphates, and was sensitive to rifampin, an antibiotic known to inhibit initiation of replication. Novobiocin and nalidixic acid also inhibited synthesis, as did the omission of ATP, N-Ethylmaleimide, an inhibitor of DNA polymerase II and III activity, but not DNA polymerase I activity, also partially inhibited the synthetic reaction, as did chloramphenicol. The plasmid DNA synthetic product was analyzed by alkaline sucrose and dye-CsCl gradient centrifugation, as well as by agarose gel electrophoresis. In each case, the product consisted of parental and intermediate forms of plasmid DNA. Some chromosomal DNA was also synthesized by a contaminating bacterial DNA-membrane complex, but this synthesis was rifampin insensitive and could be separated from plasmid DNA synthesis.     

Effect of polynucleotide adsorption on characteristics of a planar phosphatidylcholine bilayer membrane

Interaction of DNA with planar bilayer phosphatidylcholine membrane in the presence of CaCl2 increases electric conductance of the membrane several times as a result of the formation of DNA-membrane complex. The same effect was observed in the cases of ribosomal RNA and synthetic homopolymers polyA, polyU and polyA X polyU double helix.     

Effects of temperature-sensitive variants of the Bacillus subtilis dnaB gene on the replication of a low-copy-number plasmid.

The dnaB gene of Bacillus subtilis is involved in the initiation of DNA replication and also in the binding of the chromosomal origin to the bacterial membrane. We studied the effect of temperature-sensitive dnaB mutants (dnaB1 and dnaB19) on the replication and on the DNA-membrane binding of the plasmid pKW1, which was derived from the low-copy-number plasmid pBS2. In the dnaB19 mutant, pKW1 was not able to replicate at the restrictive temperature. In the dnaB1 mutant, however, the dimeric form of pKW1 DNA was preferentially produced as the restrictive temperature, but the replication of the monomeric form was totally blocked. We also examined the effects of the dnaB(Ts) gene on the DNA-membrane binding of both the double-stranded and single-stranded DNA from pKW1. The single-stranded DNA from pKW1 was prepared from the DNA of the phage M13 mp19, which contained the origin of replication of pKW1. In the dnaB1 mutant, pKW1 DNA in both the double-stranded and single-stranded form was released from the membrane at the restrictive temperature. On the other hand, in the dnaB19 mutant, only double-stranded DNA, and not single-stranded DNA, was released from the membrane at the restrictive temperature. These results suggest that the product of the dnaB gene has at least two domains which influence the replication of DNA and the binding of DNA to the cell membrane in separate ways.     

The synthesis of DNA by DNA-membrane complexes from irradiated E. coli B/r and Bs-1: the role of the membrane.

DNA-membrane complexes were isolated from lysed E. coli B/r and Bs-1, either by low g forces from a low salt solution, or by high g forces through a discontinuous sucrose gradient. The latter method was more gentle. Irradiation of the intact bacteria had no effect on the membrane macromolecules or on RNA components of these complexes. DNA loss was not significant after irradiation under anoxic conditions but complexes isolated from from Bs-1 irradiated in air showed an appreciable decrease in DNA content. In the presence of the appropriate nucleotide mixture, both 'free' DNA, found in the supernatant fractions, and rapidly sedimented membrane-associated DNA were able to synthesize DNA in the absence of added polymerase. DNA synthesis associated with 'free' DNA was more sensitive to radiation than that associated with DNA bound to the membrane, which appeared to moderate the effects of radiation on new DNA synthesis. It is concluded that the depression of DNA synthesis is primarily a result of irradiation-induced changes on genome-DNA. The interpretation of earlier work from our laboratories that DNA-membrane complexes contained the macromolecular structure which responded to radiation with a high o.e.r. is not supported by the evidence in this work.     

Studies on an Endonuclease Activity Associated with the Bacteriophage T7 DNA-Membrane Complex

Infection of Escherichia coli with bacteriophage T7 results in the formation of an endonuclease which is selectively associated with the T7 DNA-membrane complex. A specificity of association with the complex is indicated by the finding that the enzyme is completely resolved from a previously described T7 endonuclease I. When membrane complexes containing (3)H-labeled in vivo synthesized DNA are incubated in the standard reaction mixture a specific cleavage product is formed which is about one-fourth the size of T7 DNA. The endonuclease associated with the complex produces a similar cleavage product after extensive incubation with native T7 DNA or T7 concatemers. Degradation of concatemers occurs by a mechanism in which the DNA is converted to molecules one-half the size of T7. This product is in turn converted to fragments one-fourth the size of mature phage DNA. The endonuclease is not present in membrane complexes from uninfected cells or cells infected with gene 1 mutants. The enzyme activity is, however, present in cells infected with mutants defective in T7 DNA synthesis or maturation.     

Intracellular organization of bacteriophage T7 DNA: analysis of parenteral bacteriophage T7 DNA-membrane and DNA-protein complexes.

After infection of Escherichia coli with bacteriophage T7, the parenteral DNA forms a stable association with host cell membranes. The DNA-membrane complex isolated in cesium chloride gradients is free of host DNA and the bulk of T7 RNA. The complex purified through two cesium chloride gradients contains a reproducible set of proteins which are enriched in polypeptides having molecular weights of 54,000, 34,000, and 32,000. All proteins present in the complex are derived from host membranes. Treatment of the complex with Bruij-58 removes 95% of the membrane lipid and selectively releases certain protein components. The Brij-treated complex has an S value of about 1,000 and the sedimentation rate of this material is not altered by treatment with Pronase or RNase.     

Intracellular location of NRl-plasmid DNA inEscherichia coli minicells

Intracellular location of plasmid NR1 (M = 58 Mg/mol, stringent control of replication, 1–2 copies perEscherichia coli chromosomal equivalent) was studied and compared with that of plasmid R6KΔ1 (M = 21 Mg/mol, relaxed control of replication, 10–15 copies perE. coli chromosomal equivalent), both inE. coli minicells. Considerable difference in relative distribution of molecules of these two plasmid DNA’s between the cytoplasm and the membrane fraction was found when components of the corresponding minicell lyzates were fractionated by sedimentation in a double-linear gradient of caesium chlorid and sucrose. Also the difference in relative numbers of NR1 DNA and R6KΔ1 DNA molecules stably associated with the membrane of minicells, determined by electron-microscopic examination of the fractions containing plasmid DNA-membrane complexes, was evaluated as statistically significant. The association of NR1 DNA molecules withE. coli minicell membrane was found to be a much more frequent event than such association of R6KΔ1 molecules. The absolute amount of plasmid DNA associated with membrane in a single minicell corresponds to one molecule for both NR1 and R6KAΔ1.     

Deoxyribonucleic Acid-Membrane Complexes in the Bacillus subtilis Transformation System

The fate of transforming deoxyribonucleic acid (DNA) in Bacillus subtilis was studied by isolating the DNA-membrane complex on Renografin gradients. Soon after uptake, transforming DNA binds to the cell membrane and displays a greater resistance to shear than the recipient genome-membrane complex. Ten minutes after uptake, a portion of the donor DNA is released from the membrane. Most of the released donor radioactivity represents unintegrated, biologically inactive DNA. Recombinant or integrated DNA is enriched 1.5- to 1.7-fold in the membrane. This enrichment last at least 30 min after termination of DNA uptake, and probably much longer. The data suggest that transforming DNA may be integrated into the recipient genome on, or close to, the cell membrane.     

Membrane particles from Escherichia coli and Bacillus subtilis, containing penicillin-binding proteins and enriched for chromosomal-origin DNA.

Rapid-sedimenting DNA-membrane complexes were obtained from both Bacillus subtilis and Escherichia coli by a method involving gentle lysis followed by restriction enzyme digestion and sucrose gradient fractionation. These complexes were substantially enriched in chromosomal origin DNA, and in B. subtilis, the complexes were enriched in penicillin-binding proteins relative to that of the total membrane. Such complexes may represent procaryotic membrane domains which are topographically and functionally distinct.     

Nuclear membranes from mammalian liver : IV. Characterization of membrane-attached DNA

DNA associated with nuclear membranes isolated from liver tissue of mice and rats (sucklings and partially hepatectomized adults) has been analysed and directly demonstrated by electron microscopy using spreading techniques. The sensitivity of this DNA-membrane association to DNAse, to 4 M CsCl-centrifugation, urea, and to detergent has been examined and compared with that of ‘microsomal DNA’. The DNA has been purified from nuclear membrane fractions, and the purity and molecular size distribution of the preparations has been determined. The characteristics of this DNA with respect to buoyant density, melting behaviour, content of repetitive sequences, nucleotide composition, molecular configuration, and turnover and labelling kinetics with various precursors (thymidine, deoxycytidine, phosphate) have been examined and compared with the corresponding properties of DNA from whole nuclei and other nuclear subfractions. Most properties of membrane DNA are identical or similar to those of bulk nuclear DNA. It is, however, enriched in satelite DNA and other repetitive sequences to a moderate extent and differs from it in its replication rate and time. The results reflect the close relationship between the nuclear envelope and (constitutive) heterochromatin, but also indicate that membrane binding is not restricted to this material. The data speak against a preferential localization of replicating points in the nuclear membrane DNA, as well as against an initiation of replication at the nuclear envelope.     

Biosynthesis of edeine: II. Localization of edeine synthetase within Bacillus brevis Vm4.

Edeine-synthesizing polyenzymes, associated with a complex of sytoplasmic membrane and DNA, were obtained from gently lysed cells of Bacillus brevis Vm4. The polyenzymes-membrane-DNA complex, isolated from dells intensively synthesizing edeines (18--20 h culture) contained edeine B. Edeine B was found to be bound covalently t o the edeine synthetase. The amount of edeine bound to polyenzymes was 0.1--0.3 mumol/mg protein, depending on the age of cells. Detachment of deeine synthetase with a covalently bound edeine B from the membrane-DNA complex was accomplished by a treatment with (NH4)2-SO4 at 45--55% saturation or by DEAE-cellulose column fractionation. In contrast to other components of the complex, the edeine-polyenzymes fragment was not adsorbed to the DEAE-cellulose. Sephadex G-200 column chromatography separated the edeine-polyenzymes complex into 3 fractions. Edeine-polyenzymes complex, obtained from lysozyme-Brij-58-DNAase treated cells, contained edeine B bound to two protein fractions of mol. wt 210 000 and 160 000. Edeine-polyenzymes complex detached from the complex with the membrane and DNA contained edeine B, bound only to protein fraction of mol. wt 210 000. Edeine A was not found in the edeine-polyenzymes complex. No accumulation of free antibiotics within 16--22 h old cells of B. brevis Vm4 was detected. The edeine-polyenzymes complex associated with the DNA-membrane complex has shown no antimicrobial activity. By treating of above with alkali, edeine B of specific activity: 80 units/mjmol was released. The complex of DNA-membrane associated with edeine-polyenzymes complex was able to synthesize DNA, under the conditions described for synthesis, directed by a DNA-membrane complex. Edeine when associated with this complex did not effect the DNA-synthesizing activity.     

Binding of NR1 plasmid DNA to membrane during replication in Escherichia coli mini-cells

Studying the replication of NR1 plasmid in E. coli mini-cells it was shown that the character of bond between plasmid DNA and membrane depends on the stage of replication cycle of the plasmid. On initiation the DNA-membrane complex is sensitive to the action of ionic force. In the process of elongation the bond of DNA molecules with the membrane is unstable if exists at all, and can be broken even by the nonionic detergent. At the final stage of replication the newly synthesized molecules form a complex with the membrane structures which is unstable in the presence of 0,5 M NaCl. The destruction of the complex followed by the open cycle of plasmid DNA coming out of it takes place under the action of ionic detergent.     

Synthesis of nucleic acid probes on membrane supports: a procedure for the removal of unincorporated precursors

We have used DNA bound to small pieces of nylon membrane for the synthesis of radioactive probes. The DNA to be used for generating the probe(s) is first bound to nylon membranes and then introduced into the reaction mix. The labeling reaction takes place on the membrane and therefore allows easy removal of unincorporated precursors by simple washing for 1-2 min. The clean labeled probe is eluted from the membrane in formamide or in water and is ready for use. This DNA-membrane can be stored for reuse. Synthesis of probes on a solid support such as nylon membrane thus circumvents problems associated with chromatographic manipulations needed for the separation of labeled DNA from unicorporated precursors. Probes synthesized in this manner are as efficient in detecting nucleic acid sequences as those synthesized in solution.