Properties of the high-molecular-weight deoxyribonuclease of Bacillus subtilis degrading single-stranded DNA.

We have studied the properties of the high-Mr DNAse degrading single-stranded DNA which is present in extracts of Bacillus subtilis. This enzyme is a heterogeneous aggregate of identical subunits with an Mr of 36 000, as measured in dodecylsulfate/polyacrylamide electrophoresis. The aggregate can be disassembled by the presence of Triton X-100, but reforms spontaneously following removal of the detergent. A mild proteolytic treatment of the aggregate causes the irreversible and nearly quantitative conversion into the free subunit. The modified subunit has identical properties (in terms of size, chromatographic adsorption and catalytic activity) as the small DNAse previously described by Ciarrocchi et al. [Eur. J. Biochem. 61, 487 (1976)], i.e. an endonuclease highly specific for single-stranded DNA and producing 5'-P and 3'-OH ends.     

Properties of Bacillus subtilis ATP-dependent deoxyribonuclease.

A purification procedure described previously resulting in electrophoretically pure Bacillus subtilis ATP-dependent DNAse has now been modified by adding a fractionation stage with Polymin P to permit large-scale isolation of the enzyme. It has been found that the enzyme molecule (Mr = 300000) consists of two large subunits with Mr 155000 and 140000. The purified enzyme has three activities: (1) DNAse on linear single-stranded and double-stranded DNAs (2) DNA-unwinding and (3) ATPase. Circular DNAs were not affected by the enzyme. Study of the dependence of these activities on temperature, pH, and ATP and Mg2+ concentrations has revealed two different states of the enzyme. At low ATP concentrations and alkaline pH, it showed chiefly nuclease action, degrading considerable amounts of DNA to small fragments five residues long on average. At higher ATP concentrations and neutral pH (more physiological conditions) it predominantly unwound DNA. Simultaneously it cut preferentially one of the duplex strands to fragments more than 1000 residues in length. The results obtained suggest that the energy of the enzyme-cleaved ATP is mainly expended on unwinding rather than on degrading DNA molecules.     

Effect of Nalidixic Acid on Deoxyribonucleic Acid Synthesis in Bacteriophage SPO1-infected Bacillus subtilis

The effect of nalidixic acid on deoxyribonucleic acid (DNA) synthesis in Bacillus subtilis cells infected with bacteriophage SPO1 was studied. Nalidixic acid had little inhibitory effect on SPO1 DNA synthesis at concentrations that drastically inhibited B. subtilis DNA synthesis. Inhibition of DNA synthesis, appropriate to the concentration used, was imposed within 1 min after addition of nalidixic acid, suggesting that it acts directly on DNA synthesis in both infected and uninfected cells. The SPO1 DNA synthesized in the presence of high concentrations of nalidixic acid had a density characteristic of normal SPO1 DNA and was packaged into viable progeny phage particles, but its rate of synthesis was reduced and bacterial lysis was delayed.     

Effect of Nalidixic Acid on PBS2 Bacteriophage Infection of Bacillus subtilis

Infection of Bacillus subtilis by PBS2 phage, whose DNA contains uracil instead of thymine, is relatively unaffected by low concentrations of nalidixic acid which severely inhibit B. subtilis DNA synthesis. High concentrations of nalidixic acid do inhibit PBS2 DNA synthesis, but more severely reduce the burst size of PBS2 infections. Hydroxyurea blocks PBS2 DNA synthesis, preventing progeny phage production.     

The effect of parabens on DNA, RNA and protein synthesis in Escherichia coli and Bacillus subtilis

The effect of methyl, propyl and butyl esters of p-hydroxybenzoic acid on DNA and RNA synthesis has been tested in toluenized cells of Escherichia coli and Bacillus subtilis. Both RNA and DNA synthesis of these bacteria were inhibited. The inhibitory concentrations were higher than those previously reported for growth inhibition. Protein synthesis in cell-free extracts (S-30 fraction) of B. subtilis was even more sensitive to parabens than DNA and RNA synthesis, while protein synthesis in Esch. coli was largely unaffected.     

The effect of parabens on DNA, RNA and protein synthesis in Escherichia coli and Bacillus subtilis

The effect of methyl, propyl and butyl esters of p -hydroxybenzoic acid on DNA and RNA synthesis has been tested in toluenized cells of Escherichia coli and Bacillus subtilis. Both RNA and DNA synthesis of these bacteria were inhibited. The inhibitory concentrations were higher than those previously reported for growth inhibition. Protein synthesis in cell-free extracts (S-30 fraction) of B. subtilis was even more sensitive to parabens than DNA and RNA synthesis, while protein synthesis in Esch. coli was largely unaffected.     

Effect of antibody excess on the size, stoichiometry, and DNAse resistance of DNA anti-DNA immune complexes

The binding of antibodies to DNA was examined under conditions of increasing antibody excess. DNA anti-DNA immune complexes (IC) formed at increasing antibody to DNA ratios were digested with excess DNAse I, and the DNAse-resistant (protected) IC were analyzed. With increasing antibody excess, the size of the IC that were resistant to DNAse digestion increased, and the size of the protected DNA within the IC also increased. This suggested that IgG molecules could bind in close proximity along the DNA molecule, preventing access of DNAse to the DNA between adjacent IgG. To further define the binding of adjacent IgG, DNAse digested IC containing one or two IgG were isolated, and the DNA contained within these IC was analyzed on DNA sequencing gels. Binding of a single IgG to DNA resulted in the protection of a DNA fragment 35 to 45 base pairs (bp) long, corresponding to the distance between binding sites of a single IgG molecule. Binding of two IgG to DNA protected a DNA fragment 50 to 60 bp long, 1 1/2 times the size of the fragment protected by one IgG. These data suggest that in conditions of Ab excess, IgG molecules can interdigitate along the DNA molecule, resulting in small, stable, DNAse-resistant IC of high antibody density.     

Effect of liver chromatin DNAse on closed circular DNA of PM-2 phage and simian virus 40]

Comparative effect of the DNAse from rat liver chromatin and Neurospora crassa endonuclease S1 on closed circular superhelical DNA of PM-2 phage and Simian Virus 40 is studied. It is shown that both of them--the DNAse from chromatin proteins and endonuclease S1--are specific to single-stranded regions in DNA molecular. It is suggested that chromatin protein DNAse participates in reparation processes.     

Depression by NAD of x-ray-induced repair-type DNA synthesis in toluene-treated Bacillus subtilis.

NAD prevents a DNA repair-type synthesis that is dependent on polymerase I in toluene-treated, X-irradiated Bacillus subtilis. In unirradiated preparations, NAD had little effect on an ATP-dependent, semiconservative synthesis but partially inhibited a repair-type synthesis. In a mutant lacking polymerase I (polA1-), the presence of NAD did not affect dTTP utilization in DNA synthesis. Nicotinamide mononucleotide (NMN) partially reverses the NAD inhibition of repair-type DNA synthesis. NADP and FAD were ineffective as substitutes for NAD. Since NAD is the cofactor for polynucleotide ligase in Bacillus subtilis and NMN is known to discharge AMP from the active AMP ligase complex, it is proposed that activation of DNA ligase reduces dTMP incorporation by reducing sites for, or limiting DNA polymerase I action.     

DNA-tropic inhibitors of nuclease action on DNA in liver nuclei

Distamycin is a potent, wide-spectrum inhibitor of the breaking of both free and intranuclear DNA with DNAse I and with own nuclear nucleases. It compares very favourably with actinomycin D, proflavin and ethidium bromide, especially in the inhibition of DNAse I action in the nuclei. This seems likely to be due to partial overlapping of the binding sites of the nuclei with chromatin proteins in contrast to distamycin that interacts with a minor furrow of DNA being blocked to a less extent by proteins. The DNA-tropic agents under test exert no qualitative effect on the kinetics of intranuclear DNA splitting by DNAse I. Carminomycin and bleomycin are the least effective inhibitors in all the systems depicted.     

An octakis-intercalating molecule

Herein we report the synthesis and characterization of a polyintercalator with eight potential intercalating l,4,5,8-naphthalenetetracarboxylic diimide (NDI) units linked in a head-to-tail arrangement via a peptide linker. UV spectroscopy and viscometry measurements indicated the molecule binds to double-stranded DNA with all eight NDI units intercalated simultaneously. Competition dialysis and DNAse 1 footprinting studies revealed a preference for GC-rich regions of DNA, and circular dichroism studies revealed significant distortion of B-form DNA upon binding. Our so-called "octamer" represents, to the best of our knowledge, the first intercalator that binds as an octakis-intercalator, capable of spanning at least 16 base pairs of DNA.     

Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation.

A hallmark of apoptosis is the fragmentation of nuclear DNA. Although this activity involves the caspase-3-dependent DNAse CAD (caspase-activated DNAse), evidence exists that DNA fragmentation can occur independently of caspase activity. Here we report on the ability of truncated Bid (tBid) to induce the release of a DNAse activity from mitochondria. This DNAse activity was identified by mass spectrometry as endonuclease G, an abundant 30 kDa protein released from mitochondria under apoptotic conditions. No tBid-induced endonuclease G release could be observed in mitochondria from Bcl-2-transgenic mice. The in vivo occurrence of endonuclease G release from mitochondria during apoptosis was confirmed in the liver from mice injected with agonistic anti-Fas antibody and is completely prevented in Bcl-2 transgenic mice. These data indicate that endonuclease G may be involved in CAD-independent DNA fragmentation during cell death pathways in which truncated Bid is generated.     

Formation of additional contacts of chromosome with membrane in the process of DNA repair synthesis in bacterial cells

An increase in the amount of membrane-bound DNA was found in B. subtilis cells with UV-induced DNA repair synthesis as compared to untreated cells. It was shown that DNA repair synthesis occurred in DNA membrane complexes (DMC) formed during UV-irradiation. UV-induced formation of DMC was observed in cells of wild type strains which were capable of repairing damaged DNA but not in a mutant defective in DNA-polymerase I. It was demonstrated that DNA-polymerase I is located on the membrane of B. subtilis cells. This suggested a participation of DNA-polymerase I in binding of the chromosome to the membrane in UV-irradiated cells. UV-induced DMC did not dissociate when the cells were treated with inhibitors of DNA-gyrase. It, therefore, was qualitatively different from the DMC found during replication. The mechanisms of binding of the damaged DNA to the membrane in UV-irradiated cells of B. subtilis are discussed.     

Regulation of synthesis of alkaline phosphatase by deoxyribonucleic acid synthesis in a constitutive mutant of Bacillus subtilis

Hiraga, Sota (Osaka University, Osaka, Japan). Regulation of synthesis of alkaline phosphatase by deoxyribonucleic acid synthesis in a constitutive mutant of Bacillus subtilis. J. Bacteriol. 91:2192-2199. 1966.-It was found that synthesis of alkaline phosphatase (APase) correlated with deoxyribonucleic acid (DNA) synthesis in a partially constitutive mutant of Bacillus subtilis. When cultures of the mutant were made to undergo synchronous growth by germination of spores in an excess-phosphate medium, synthesis of APase was repressed at the beginning of DNA synthesis. If the initiation of DNA synthesis was inhibited by thymine starvation, the repression of APase was not observed. When DNA synthesis, previously initiated, was inhibited by thymine or uracil starvation, or by addition of mitomycin C, the repression was partially released at a later stage. In contrast, this correlation between repression and DNA synthesis was not observed in a repressible strain.     

Electron immunohistochemical analysis of localization of neutral Mn2+-dependent DNAase. III. Visualization of DNAse binding to isolated chromatin

Neutral Mn(2+)-dependent DNAse is localized on isolated chromatin structures in both normal and regenerating rat liver. The enzyme was revealed located along the whole length of nucleosomal chain and in hypernucleosomal structures. However, as concerns the quantity of the enzyme, it was distributed unevently along the chromatin, thus reflecting the pattern of different functional states of native chromatin. According to biochemical and immunohistochemical data, DNAse can hydrolyse in vitro only one-stranded DNA. One of possible explanations of the observed differences in DNAse binding with native DNA chromatin and its inability to adsorb on native DNA in vitro may be the presence of hypothetical DNA-binding proteins in native chromatin making complexes with DNAse and thereby responsible for immobilization of the enzyme on chromatin structures in vivo.     

Blow fly Lucilia sericata nuclease digests DNA associated with wound slough/eschar and with Pseudomonas aeruginosa biofilm

In chronic wounds, it may be clinically important to remove extracellular bacterial and patient DNA as its presence may impede wound healing and promote bacterial survival in biofilm, in which extracellular DNA forms part of the biofilm architecture. As medicinal maggots, larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) have been shown to efficiently debride wounds it became of interest to investigate their excretions/secretions (ES) for the presence of a deoxyribonuclease (DNAse) activity. Excretions/secretions products were shown to contain a DNAse, with magnesium, sodium and calcium metal ion dependency, and a native molecular mass following affinity purification of approximately 45 kDa. The affinity purified DNAse degraded genomic bacterial DNA per se, DNA from the slough/eschar of a venous leg ulcer, and extracellular bacterial DNA in biofilms pre‐formed from a clinical isolate of Pseudomonas aeruginosa. The latter finding highlights an important attribute of the DNAse, given the frequency of P. aeruginosa infection in non‐healing wounds and the fact that P. aeruginosa virulence factors can be toxic to maggots. Maggot DNAse is thus a competent enzyme derived from a rational source, with the potential to assist in clinical wound debridement by removing extracellular DNA from tissue and biofilm, and promoting tissue viability, while liberating proteinaceous slough/eschar for debridement by the suite of proteinases secreted by L. sericata.     

Initiation of Bacillus subtilis Ribonucleic Acid Polymerase on Deoxyribonucleic Acid from Bacteriophages 2C, φ29, T4, and Lambda

Ribonucleic acid (RNA) synthesis primed by bacteriophage T4 or lambda deoxyribonucleic acid (DNA) with Bacillus subtilis RNA polymerase is severely inhibited by high ionic strength. In contrast, RNA synthesis on B. subtilis bacteriophage 2C, SPO1, or phi29 DNA is only moderately affected under similar conditions. The basis of this inhibition lies in the inability of the enzyme to initiate RNA chains with adenosine triphosphate or guanosine triphosphate (ATP, GTP). Binding to templates and the rate of catalysis in high salt after initiation do not seem to be affected. Incorporation of gamma-(32)P-ATP and GTP under a variety of conditions suggests that the specificity of B. subtilis RNA polymerase is different from that of the Escherichia coli enzyme and that it recognizes few promoters on T4 and lambda DNA. Although B. subtilis RNA polymerase initiates RNA chains primarily with ATP or GTP, initiations with pyrimidines can occur on DNA molecules in which hydroxymethyluracil replaces thymine. RNA synthesis on denatured DNA does not seem to be inhibited by high ionic strength, and on native T4 or lambda DNA the inhibition of initiation at constant ionic strength is inversely but not linearly proportional to the ionic radii of cations used to stabilize bihelical DNA to denaturation.     

Effect of nalidixic acid on the activation of RNA synthesis in outgrowing spores of Bacillus subtilis

Outgrowth of B. subtilis spores depends on the action of DNA gyrase (comp. Matsuda and Kameyama 1980). Application of nalidixic acid (100 micrograms/ml) to dormant spores of Bacillus subtilis prevents the outgrowth. Application of nalidixic acid (100 micrograms/ml) during the early outgrowth phase (after a 20 min germination period) does not prevent, but only delay spore outgrowth. Germination of spores is not influenced. Nalidixic acid is an effective inhibitor of RNA synthesis in outgrowing spores, whereas vegetative cells are more resistant. Spores can grow out inspite of a remarkably reduced intensity of RNA synthesis. Nalidixic acid particularly inhibits the synthesis of stable RNA, probably that of ribosomal RNA. We suggest that DNA gyrase-catalyzed alterations in DNA structure are involved in the regulation of the gene expressional program of outgrowing B. subtilis spores.     

The sperm nuclear matrix is required for paternal DNA replication

The mammalian sperm nucleus provides an excellent model for studying the relationship between the formation of nuclear structure and the initiation of DNA replication. We previously demonstrated that mammalian sperm nuclei contain a nuclear matrix that organizes the DNA into loop domains in a manner similar to that of somatic cells. In this study, we tested the minimal components of the sperm nucleus that are necessary for the formation of the male pronucleus and for the initiation of DNA synthesis. We extracted mouse sperm nuclei with high salt and dithiothreitol to remove the protamines in order to form nuclear halos. These were then treated with either restriction endonucleases to release the DNA not directly associated with the nuclear matrix or with DNAse I to digest all the DNA. The treated sperm nuclei were injected into oocytes, and the paternal pronuclear formation and DNA synthesis was monitored. We found that restriction digested sperm nuclear halos were capable of forming paternal pronuclei and initiating DNA synthesis. However, when isolated mouse sperm DNA or sperm DNA reconstituted with the nuclear matrices were injected into oocytes, no paternal pronuclear formation or DNA synthesis was observed. These data suggest that the in situ nuclear matrix attachment organization of sperm DNA is required for mouse paternal pronuclear DNA synthesis.     

The influence of the exocyclic pyrimidine 5-methyl group on DNAse I cleavage and sequence recognition by drugs

Incorporation of modified nucleotides into DNA, using the PCR, has allowed us to probe the influence that the exocyclic 5-methyl group of pyrimidines has on DNAse I cleavage and sequence recognition by drugs. The results show that removal of the methyl group from the major groove, made possible by substituting uridine for thymidine, allows DNAse I to cleave more readily at AT-rich regions compared to normal DNA. By contrast, addition of an extra methyl group, contrived by substituting 5-methylcytidine for normal cytidine, allows DNAse I to cleave more readily at GC-rich regions compared to normal DNA. In the cutting pattern of DNA containing both uridine and 5-methyl cytosine, we find the cleavage characteristics of both the single-substituted DNA species combined. Thus, the presence or absence of the exocyclic 5-methyl group in the major groove has a strong influence on the relative intensity of cleavage of phosphodiester bonds by DNAse I. These nucleotide substitutions can also influence the sequence-selective binding of drugs to DNA. Whereas removal of the methyl group (replacement of T with U) generally has little effect on sequence recognition by a variety of drugs, addition of a methyl group (replacement of C with M) generates new binding sites for some intercalators, namely daunomycin, DACA and SN16713.