Induction of intracellular endonuclease synthesis in Serratia marcescens by agents suppressing DNA replication]

Endonuclease synthesis in Serratia marcescens was studied in the presence of agents selectively suppressing DNA biosynthesis: nalidixic acid, mitomycin, hydroxyurea and thymine limitation. All the agents suppressing DNA replication induced exocellular endonuclease biosynthesis irrespective of their action mechanism. The greatest inducing effect was exerted when the agents were added to cells in the late exponential phase. Endonuclease biosynthesis was induced 1-2 hours after adding the agent and was inhibited with chloramphenicol. The induction of exocellular endonuclease synthesis in Serratia marcescens by the classical inducing agents of a SOS response seems to be indicative of a Lex A regulated process.     

SOS-like induction in Bacillus subtilis: induction of the RecA protein analog and a damage-inducible operon by DNA damage in Rec+ and DNA repair-deficient strains.

We quantitated the induction of the Bacillus subtilis Rec protein (the analog of Escherichia coli RecA protein) and the B. subtilis din-22 operon (representative of a set of DNA damage-inducible operons in B. subtilis) following DNA damage in Rec+ and DNA repair-deficient strains. After exposure to mitomycin C or UV irradiation, each of four distinct rec (recA1, recB2, recE4, and recM13) mutations reduced to the same extent the rates of both Rec protein induction (determined by densitometric scanning of immunoblot transfers) and din-22 operon induction (determined by assaying beta-galactosidase activity in din-22::Tn917-lacZ fusion strains). The induction deficiencies in recA1 and recE4 strains were partially complemented by the E. coli RecA protein, which was expressed on a plasmid in B. subtilis; the E. coli RecA protein had no effect on either induction event in Rec+, recB2, or recM13 strains. These results suggest that (i) the expression of both the B. subtilis Rec protein and the din-22 operon share a common regulatory component, (ii) the recA1 and recE4 mutations affect the regulation and/or activity of the B. subtilis Rec protein, and (iii) an SOS regulatory system like the E. coli system is highly conserved in B. subtilis. We also showed that the basal level of B. subtilis Rec protein is about 4,500 molecules per cell and that maximum induction by DNA damage causes an approximately fivefold increase in the rate of Rec protein accumulation.     

Integration and Induction of Phage P22 in a Recombination-deficient Mutant of Salmonella typhimurium

Phage P22 can integrate as prophage into a recombination-deficient (Rec(-)) strain of Salmonella typhimurium. At 37 C, the integration efficiency is only 10% that in Rec(+) infection, but at 25 C the efficiencies in Rec(-) and Rec(+) hosts are similar. Rec(-) lysogens cannot be induced by ultraviolet irradiation or by treatments with the chemical inducing agents streptonigrin or mitomycin C. Heat induction of Rec(-) cells lysogenic for a temperature-sensitive c(2) mutant (ts c(2)) is normal, showing that the Rec(-) cell has the machinery necessary for prophage excision. Ultraviolet irradiation of Rec(-) (ts c(2)) lysogens prior to heat induction does not prevent the formation of infective centers after temperature shift. Thus, the noninducibility of Rec(-) lysogens is not due to destruction of the prophage as a result of ultraviolet irradiation. Deoxyribonucleic acid-ribonucleic acid (RNA) hybridization experiments demonstrate that no increase in phage-specific RNA synthesis occurs after ultraviolet irradiation of a Rec(-) (c(+)) lysogen. The Rec(-) mutant appears to lack part of the mechanism required to destroy the phage repressor and allow the initiation of early phage functions such as messenger RNA synthesis. A similar conclusion was reached previously for an Escherichia coli Rec(-) strain.     

Induction of theEscherichia coli UVM response by oxidative stress

UVM (ultravioletmodulation of mutagenesis) is a recently describedrecA-independent, inducible mutagenic phenomenon in which prior UV irradiation ofEscherichia coli cells strongly enhances mutation fixation at a site-specific 3-N⁴-ethenocytosine (C) lesion borne on a transfected single-stranded M13 DNA vector. Subsequent studies demonstrated that UVM is also induced by alkylating agents, and is distinct from both the SOS response and the adaptive response to alkylation damage. Because of the increasing significance being attributed to oxidative DNA damage, it is interesting to ask whether this class of DNA damage can also induce UVM. By transfecting M13 vector DNA bearing a site-specificC lesion into cells pretreated with inducing agents, we show here that the oxidative agent H₂O₂ is a potent inducer of UVM, and that the induction of UVM by H₂O₂ does not requireoxyR-regulated gene expression. UVM induction by H₂O₂ appears to be mediated by DNA damage, as indicated by the observation of a concomitant reduction in cellular toxicity and UVM response in OxyR^c cells. Available evidence suggests that UVM represents a generalized cellular response to a broad range of chemical and physical genotoxicants, and that DNA damage constitutes the most likely signal for its induction.     

Regulation of the SOS response in Bacillus subtilis: evidence for a LexA repressor homolog.

The inducible SOS response for DNA repair and mutagenesis in the bacterium Bacillus subtilis resembles the extensively characterized SOS system of Escherichia coli. In this report, we demonstrate that the cellular repressor of the E. coli SOS system, the LexA protein, is specifically cleaved in B. subtilis following exposure of the cells to DNA-damaging treatments that induce the SOS response. The in vivo cleavage of LexA is dependent upon the functions of the E. coli RecA protein homolog in B. subtilis (B. subtilis RecA) and results in the same two cleavage fragments as produced in E. coli cells following the induction of the SOS response. We also show that a mutant form of the E. coli RecA protein (RecA430) can partially substitute for the nonfunctional cellular RecA protein in the B. subtilis recA4 mutant, in a manner consistent with its known activities and deficiencies in E. coli. RecA430 protein, which has impaired repressor cleaving (LexA, UmuD, and bacteriophage lambda cI) functions in E.coli, partially restores genetic exchange to B. subtilis recA4 strains but, unlike wild-type E. coli RecA protein, is not capable of inducing SOS functions (expression of DNA damage-inducible [din::Tn917-lacZ] operons or RecA synthesis) in B. subtilis in response to DNA-damaging agents or those functions that normally accompany the development of physiological competence. Our results provide support for the existence of a cellular repressor in B. subtilis that is functionally homologous to the E. coli LexA repressor and suggest that the mechanism by which B. subtilis RecA protein (like RecA of E. coli) becomes activated to promote the induction of the SOS response is also conserved.     

Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication

The SOS genes of Escherichia coli, which include many DNA repair genes, are induced by DNA damage. Although the central biochemical event in induction, activation of RecA protein through binding of single-stranded DNA and ATP to promote cleavage of the LexA repressor, is known, the cellular event that provides this activation following DNA damage has not been well understood. We provide evidence here that the major pathway of induction after damage by a typical agent, ultraviolet light, requires an active replication fork; this result supports the model that DNA replication leaves gaps where elongation stops at damage-induced lesions, and thus provides the single-stranded DNA that activates RecA protein. In order to detect quantitatively the immediate product of the inducing signal, activated RecA protein, we have designed an assay to measure the rate of disappearance of intact LexA repressor. With this assay, we have studied the early phase of the induction process. LexA cleavage is detectable within minutes after DNA damage and occurs in the absence of protein synthesis. By following the reaccumulation of LexA in the cell, we detect repair of DNA and the disappearance of the inducing signal. Using this assay, we have measured the LexA content of wild-type and various mutant cells, characterized the kinetics and conditions for development of the inducing signal after various inducing treatments and, finally, have shown the requirement for DNA replication in SOS induction by ultraviolet light.     

Detection of induced synthesis of colicin E9 using ColE9p::gfpmut2 based reporter system

The majority of colicin operons are regulated by an SOS response inducible promoter (SOS promoter), located at upstream of the colicin operons. Therefore, colicin synthesis is induced by DNA damaging agents like mitomycin C (MMC) because the resulting DNA damage switches on the SOS response in bacteria. In this study, we have described the strategy for fusion of the SOS promoter of the colicin E9 operon (ColE9p) with a promoterless green fluorescent reporter gene (gfpmut2). We observed that the ColE9p–gfpmut2 is inducible by MMC which confirmed that the ColE9p–gfpmut2 is sensitive to SOS response inducing agents. The data implies that the ColE9p–gfpmut2 based reporter system is suitable for monitoring the ColE9 synthesis induced by SOS response inducing agents including antibiotics. Using green fluorescent protein expression from the ColE9p–gfpmut2 as an indicator of ColE9 synthesis; we have investigated, first time, the inducing effects of cephalexin antibiotic on ColE9 synthesis. Our data demonstrated that the cephalexin has potential to induce ColE9 synthesis from E. coli JM83 host cells albeit the level of this induction is very low hence its detection required a highly sensitive method.     

Participation of exogenous DNA in the repair processes of meristematicVicia faba cells injured by monofunctional alkylating agens ethylmethane sulphonate

The effect of exogenous DNA of syngeneic origin on the course of reparation of meristematic cells ofVicia faba primary roots followed after treatment with monofunctional alkylating agent, ethyl methanesulphonate (EMS), was tested. Time course of alternations in mitotic activity of investigated cellular population and the dynamics of formation of postmetaphase chromosomal aberrations was evaluated. A reparation of damaged cells was significantly supported by syngeneic DNA; its application induced an increased incidence of cellular division already in the early intervals of the repair which was accompanied by concomitant decrease of microscopically detectable rupture in the chromosomes. The study performed on the localization of induced damages occurring in metaphase chromosomes pointed out an increased sensitivity of small chromosomes ofVicia faba to EMS. Similarly, a reparative action of syngeneic DNA was exhibited by significant decrease of aberrations frequency, predominantly in the same chromosomal group. Per cent representation of individual types, not affected by the action of syngeneic DNA, was established by detailed classification of induced aberrations. In both cases, isochromatide breaks were found of greatest predominance.     

Molecular dosimetry for sister-chromatid exchange induction and cytotoxicity by monofunctional and bifunctional alkylating agents

The induction of sister-chromatid exchanges (SCEs) and cytotoxicity in 9L cells treated with monofunctional and bifunctional alkylating agents has been investigated. Three classes of monofunctional and bifunctional agents were studied: nitrosoureas, mustards and epoxides. Independent of class the bifunctional agents were 55–630-fold more effective at inducing SCEs and 300–2400-fold more effective at inducing cellular cytotoxicity than the corresponding monofunctional agents. Comparing the induction of SCEs and cytotoxicity by these agents showed that these two cellular responses to DNA damage are highly correlated. The extent of DNA alkylation in cells treated with 1-ethyl-1-nitrosourea (ENU) or 1-(2-chloro-ethyl)-1-nitrosourea (CNU) was similar indicating that the increased effectiveness of CNU to induce SCEs and cytotoxicity was not due to increased DNA alkylation. Molecular dosimetry calculations indicate that for CNU and ENU treatment of 9L cells there are 116 and 8500 alkylations per SCE induced and 2.6 × 10⁴ and 4.6 × 10⁶ alkylations at the dose required to reduce survival of 9L cells by 90%. Comparison of the DNA alkylation products produced by CNU and ENU treatment of 9L cells suggests that the formation of the intrastrand crosslink N⁷-bis(guanyl)ethane the interstrand crosslink 1-(3-deoxycytidyl)-2-(1-deoxyguanosinyl)ethane by CNU is responsible for the increased effectiveness of CNU treatment at both induction of SCEs and cytotoxicity.     

Nativity of the macromolecule of isologous DNA as a condition for its reparative effect on radiation damage

The effect of isologous DNA on the course of postirradiation reparation of meristematic cells ofVicia faba primary roots was studied in detail. A considerable interest was devoted to determinations of fundamental qualitative and quantitative conditions of the above effect of isologous DNA. Main criteria of the effect were both mitotic activity of irradiated cellular population and dynamics of chromosome aberrations induced by radiation. One set of experiments compared the course of reparation as occurred in regard to applied dose of ionizing radiation in native isologous DNA, DNA denaturated by heat and degraded by DNAase, and post-irradiation reparation of induced damages was favorably affected by native isologous DNA only. Another set of results evaluated the dependence of positive reparative effect of native isologous DNA on the length of the molecule demonstrating that in the process of reparation the presence of a complete DNA macromolecule was not essential. The last experimental group was focused on observations on the dependence of the rate of native isologous DNA effect on concentration of applied solution of the macromolecule.     

Induction of S.cerevisiae MAG 3-methyladenine DNA glycosylase transcript levels in response to DNA damage.

We previously showed that the expression of the Saccharomyces cerevisiae MAG 3-methyladenine (3MeA) DNA glycosylase gene, like that of the E. coli alkA 3MeA DNA glycosylase gene, is induced by alkylating agents. Here we show that the MAG induction mechanism differs from that of alkA, at least in part, because MAG mRNA levels are not only induced by alkylating agents but also by UV light and the UV-mimetic agent 4-nitroquinoline-1-oxide. Unlike some other yeast DNA-damage-inducible genes, MAG expression is not induced by heat shock. The S. cerevisiae MGT1 O6-methylguanine DNA methyltransferase is not involved in regulating MAG gene expression since MAG is efficiently induced in a methyltransferase deficient strain; similarly, MAG glycosylase deficient strains and four other methylmethane sulfonate sensitive strains were normal for alkylation-induced MAG gene expression. However, de novo protein synthesis is required to elevate MAG mRNA levels because MAG induction was abolished in the presence of cycloheximide. MAG mRNA levels were equally well induced in cycling and G1-arrested cells, suggesting that MAG induction is not simply due to a redistribution of cells into a part of the cell cycle which happens to express MAG at high levels, and that the inhibition of DNA synthesis does not act as the inducing signal.     

Genotoxicity of radiofrequency signals. I. Investigation of DNA damage and micronuclei induction in cultured human blood cells

As part of a comprehensive investigation of the potential genotoxicity of radiofrequency (RF) signals emitted by cellular telephones, in vitro studies evaluated the induction of DNA and chromosomal damage in human blood leukocytes and lymphocytes, respectively. The signals were voice modulated 837 MHz produced by an analog signal generator or by a time division multiple access (TDMA) cellular telephone, 837 MHz generated by a code division multiple access (CDMA) cellular telephone (not voice modulated), and voice modulated 1909.8 MHz generated by a global system of mobile communication (GSM)-type personal communication systems (PCS) cellular telephone. DNA damage (strand breaks/alkali labile sites) was assessed in leukocytes using the alkaline (pH>13) single cell gel electrophoresis (SCG) assay. Chromosomal damage was evaluated in lymphocytes mitogenically stimulated to divide postexposure using the cytochalasin B-binucleate cell micronucleus assay. Cells were exposed at 37+/-1 degrees C, for 3 or 24 h at average specific absorption rates (SARs) of 1.0-10.0 W/kg. Exposure for either 3 or 24 h did not induce a significant increase in DNA damage in leukocytes, nor did exposure for 3 h induce a significant increase in micronucleated cells among lymphocytes. However, exposure to each of the four RF signal technologies for 24 h at an average SAR of 5.0 or 10.0 W/kg resulted in a significant and reproducible increase in the frequency of micronucleated lymphocytes. The magnitude of the response (approximately four fold) was independent of the technology, the presence or absence of voice modulation, and the frequency (837 vs. 1909.8 MHz). This research demonstrates that, under extended exposure conditions, RF signals at an average SAR of at least 5.0 W/kg are capable of inducing chromosomal damage in human lymphocytes.     

Induction of theEscherichia coli UVM response by oxidative stress

UVM (ultravioletmodulation of mutagenesis) is a recently describedrecA-independent, inducible mutagenic phenomenon in which prior UV irradiation ofEscherichia coli cells strongly enhances mutation fixation at a site-specific 3-N⁴-ethenocytosine (?C) lesion borne on a transfected single-stranded M13 DNA vector. Subsequent studies demonstrated that UVM is also induced by alkylating agents, and is distinct from both the SOS response and the adaptive response to alkylation damage. Because of the increasing significance being attributed to oxidative DNA damage, it is interesting to ask whether this class of DNA damage can also induce UVM. By transfecting M13 vector DNA bearing a site-specific?C lesion into cells pretreated with inducing agents, we show here that the oxidative agent H₂O₂ is a potent inducer of UVM, and that the induction of UVM by H₂O₂ does not requireoxyR-regulated gene expression. UVM induction by H₂O₂ appears to be mediated by DNA damage, as indicated by the observation of a concomitant reduction in cellular toxicity and UVM response in OxyR^c cells. Available evidence suggests that UVM represents a generalized cellular response to a broad range of chemical and physical genotoxicants, and that DNA damage constitutes the most likely signal for its induction.     

Relationship Between Prophage Induction and Transformation in Haemophilus influenzae

The interaction between transformation and prophages of HP1c1, S2, and a defective phage of Haemophilus influenzae has been investigated by measurement of (i) the effect of prophage on transformation frequency and (ii) the effect of transformation on phage induction. The presence of any of the prophages does not appreciably alter transformation frequencies in various Rec(+) and Rec(-) strains. However, exposure of competent lysogens to transforming deoxyribonucleic acid (DNA) may induce phage but only in Rec(+) strains, which are able to integrate transforming DNA into their genome. Transformation of Rec(+) lysogens with DNA irradiated with ultraviolet (UV) light causes the production of even more phage than results from unirradiated DNA, but this indirect UV induction is not as effective as direct induction by UV irradiation of lysogens. Both types of UV induction are influenced by the repair capacity of the host. Wild-type cells contain a prophage and can be induced by transformation to produce a defective phage, which kills a small fraction of the cells. Defective phage in wild-type cells are also induced by H. parainfluenzae DNA, and a much larger fraction of the cells is killed. Strain BC200, which is highly transformable but is not inducible for defective phage, is not killed by H. parainfluenzae DNA, suggesting that wild-type cells are killed by killed by this DNA because of phage induction. A minicell-producing mutant, LB11, has been isolated. Some phage induction occurs in this strain when the cells are made competent, unlike the wild type. A large majority of LB11 cells surviving the competence regime are killed by exposure to transforming DNA.     

Thymineless Death in Escherichia coli 15T− and Recombinants of 15T− and Escherichia coli K-12

Thymineless death was examined in Escherichia coli 15T(-) and recombinants of 15T(-) and E. coli K-12. Those strains that were very sensitive to thymine deprivation were also very sensitive to a variety of inducing agents (mitomycin C, ultraviolet light, hydroxyurea, and nalidixic acid). Those strains that were relatively resistant to thymineless death were also relatively resistant to the inducing agents. After exposure to thymineless death and the inducing agents, sensitive strains lysed, produced colicin, and had phage particles in their lysates. These strains also showed an increase in the 6-methyladenine content of their deoxyribonucleic acid (DNA) and an increase in the DNA methylase activity of their crude extracts under these conditions. None of these effects was noted in the strains relatively resistant to thymineless death and the inducing agents. These data indicate that there are two types of thymineless death. One is represented by the strains that are very sensitive to thymine deprivation and other inducing agents and is secondary to the induction of phage psi. The strains more resistant to thymine deprivation and the other inducing agents undergo a non-phage-mediated thymineless death. The mechanism of this latter process is currently under study.     

Proprietes antibacteriennes et genotoxiques de 33 mycotoxines

Most of the 33 fungal metabolites tested provoke:

1. Bacterial growth inhibition of Bacillus thuringiensis similar to lethal effect of antibiotics.
2. Positive response in the ‘Rec’ assay using strains of Bacillus subtilis; this fact shows that these toxins are DNA modifying agents.
3. Enlargement of cell volume in the first bacteria species; this cell-abnormality induction resembles those obtained with mitomycin C.

Correlation between elongation of cells (filamentation) and in vivo carcinogenicity of mycotoxins is discussed. The filamentation should be an expression of a perturbated DNA replication (S.O.S.-error prone repair) as the consequence of DNA damages induced by genotoxic agents (i.e. carcinogens).