Mutagenic DNA repair in Escherichia coli XXI. A stable SOS-inducing signal persisting after excision repair of ultraviolet damage

Mutations to streptomycin resistance induced by ultraviolet light in Escherichia coli can lose their susceptibility to photoreversing light during excision repair and in the absence of chromosomal replication and protein synthesis, i.e., under conditions where SOS induction cannot occur. Using fusions of lac with sulA and umuC we have shown that after excision of UV damage in the presence of chloramphenicol there is a persisting, relatively stable signal capable of inducing SOS genes when protein sysnthesis is subsequently permitted. The persisting signal is formed roughly in proportion to the square of the UV dose and is about 30% photoreversible. It is suggested that the persisting SOS-inducing signal comprises a UV photoproduct (the target lesion) opposite a gap in the opposing DNA strand, and is formed by excision of one (the ancillary lesion) of a pair of closely opposed photoproducts. Calculations suggest that as few as two or three such configurations in a cell can lead to induction a sulA when protein synthesis is permitted. It is not clear whether these configurations can directly induce the SOS system because of their region of single-stranded DNA or whether the ultimate SOS-inducing signal is a more extensive single-stranded region formed when such configurations encounter a replication fork. Photoproduct/gap configurations have been previously suggested to be potentially mutagenic. UV-induced mutations to streptomycin resistance are mostly at A:T sites and are not photoreversible in fully SOS-induced bacteria in the absence of excision repair, indicating that they are not targeted at cyclobutane-type pyrimidine dimers. In SOS-induced excision-proficient bacteria there is about 39% photoreversibility which is rapidly lost after UV. This photoreversibility is attributed to many ancillary lesions being cyclobutane-type pyrimidine dimers which are excised leading to the exposure of target lesions on the opposing strand which, at these particular sites, are mostly non-photoreversible photoproducts.     

Physiological aspects of modification and restoration of chromosomal synthesis in bacteria after x-irradiation

Billen, Daniel (The University of Texas, Houston), and Roger Hewitt. Physiological aspects of modification and restoration of chromosomal synthesis in bacteria after X irradiation. J. Bacteriol. 90:1218-1225. 1965.-A study was made of the effect of amino acid deprivation or chloramphenicol on the character of postirradiation deoxyribonucleic acid (DNA) replication in bacteria with the use of radioisotopes and 5-bromouracil as a density label. CsCl density-gradient studies of DNA showed that postirradiation incubation of amino acid-requiring Escherichia coli in an amino acid-free medium interfered with continued linear chromosomal replication. In the presence of the required amino acids, linear chromosomal replication was shown to resume. Addition of chloramphenicol was found to prevent this resumption. Deletion of the required amino acids or the presence of chloramphenicol in a fully supplemented medium allowed the detection of altered DNA synthesis in bacteria at X-ray doses as low as 500 r. The character of the limited DNA made in the presence of the density label after irradiation is described. The results are interpreted as showing that the synthesis of a protein(s) is required for restoration of linear chromosomal replication in the irradiated cells.     

UV-inducible repair: influence on survival, dimer excision, DNA replication and breakdown in Escherichia coli B/r Her+ cells.

Summary Using a model of double-UV-irradiation with inducing¹ (non-lethal) and lethal fluences² we have studied involvement of UV-inducible functions in post-UV-irradiation restoration processes and survival of Escherichia coli B/r thy ^- thy ^- Hcr⁺. Cells irradiated with both inducing and lethal fluences differed from cells irradiated with lethal fluence in the following respects: They were more UV resistant; they did not die during postincubation with chloramphenicol³; they exhibited a significant reduction in dimer excision; they were able to resume DNA replication and produce normal-sized DNA molecules in the presence of chloramphenicol. Since induction was provoked in cell prestarved for amino acids it was not associated with damage to points active in replication. However, the inducible product was more important for repair of replicating than non-replicating cells. The data indicate that protein necessary for resumption of DNA synthesis after UV is not constitutive but inducible.Abbreviations ¹IF inducing fluence - ²IF lethal fluence - ³CAP chloramphenicol     

Effect of Rifampicin and Chloramphenicol on Progeny Single-stranded DNA Synthesis of Bacteriophage ϕX174

Neither bacteriophage ?X174 single-stranded DNA synthesis nor phage growth was affected by rifampicin (200 μg/ml) once it started, whereas a low concentration of chloramphenicol (30 μg/ml) inhibited the phage growth when added in a late phase of infection. When rifampicin was added at a stage where double-stranded duplex (RF) DNA replication proceeded preferentially in the presence of chloramphenicol, or even after chloramphenicol was removed before the addition of rifampicin, both single-stranded DNA synthesis and phage growth were inhibited. These results suggest that RNA synthesis sensitive to rifampicin was necessary to initiate single-stranded DNA synthesis, but no longer needed once ?X174 DNA synthesis started.     

Evidence that ultraviolet light-induced DNA replication death of recA bacteria is prevented by protein synthesis in repair-proficient bacteria

The ultraviolet light (UV) survival curve of Escherichia coli WP10 recA trp is almost biphasic, with a greatly reduced shoulder but demonstrating a transition to a decreased slope with increasing fluences, indicating the presence in the culture of a low frequency of resistant cells. Treatment of the culture with chloramphenicol before UV exposure brought almost all of the cells to a high degree of UV resistance, by bringing them to the end of their DNA replication cycle. The survival curves of the repair-proficient E. coli WP2 trp showed a similar pattern with chloramphenicol treatment or tryptophan starvation before UV exposure, but only if protein synthesis were blocked by chloramphenicol for 60 min after UV exposure. The results suggest that when recA/lexA-regulon induction is prevented, either by the recA mutation or by inhibition of protein synthesis after UV exposure, death occurs unless the cells are in the resistant state characteristic of bacteria at the end of their DNA replication cycle. With repair-proficient bacteria treated before UV exposure with chloramphenicol, when protein synthesis is not blocked after UV exposure, a marked expansion of the shoulder occurs because of the function of another resistance-conferring mechanism. This mechanism also depends on the recA+ gene since expansion of the shoulder does not occur in recA bacteria when protein synthesis is inhibited before UV exposure.     

Initiation of chromosome replication in Escherichia coli. I. Requirements for RNA and protein synthesis at different growth rates

The effects of inhibition of protein and RNA synthesis on initiation of chromosome replication in Escherichia coli$\text{B}\text{r}$ were determined by measuring rates of DNA synthesis during the division cycle before and after addition of chloramphenicol and rifampicin. The ability of cells to initiate a round of replication depended upon the pattern of chromosome replication during the division cycle. Initiation in the presence of chloramphenicol (200 μ/ml) and rifampicin (100 gmg/ml) was observed only in slowly growing cells which normally initiated a new round between the end of the previous round and the subsequent division (i.e. in the D period of the division cycle). The cells that initiated were in the D period at the time of addition of the drugs. Rapidly growing cells which normally initiated before the D period and slowly growing cells which normally initiated after the D period did not initiate in the presence of the drugs. The contrasting effects of the drugs in cells possessing different chromosome replication patterns, and the coupling between septum-crosswall formation (the D period) and initiation suggest that the timing of initiation of chromosome replication in E. coli is controlled by the cell envelope.     

Induction of mutations inSerratia marcescens by a proteosynthesis block

The formation of colour mutations ofSerratia marcescens by the action of chloramphenicol was studied. Pink variants were the commonest; the proportion of white variants was much smaller. Almost 100% mutations were formed in a two-day culture containing 100 μg. chloramphenicol/ml. Comparative experiments showed that the change in pigment formation was hereditary, i.e. that actual mutation, and not selection of chloramphenicol-resistant mutants, occurred. Mutation occurred both in strain 151 and strain HY. The resultant mutations remained constant throughout ten passages on normal nutrient medium. The minimum chloramphenicol concentration which produced an increase in the mutation frequency was 5 μg./ml. The combined effect of X-ray irradiation and chloramphenicol treatment somewhat stimulated the increase in the frequency of mutation as compared with cells which were only irradiated. The increase in the frequency of mutation was much slower on solid medium containing chloramphenicol.     

Deoxyribonucleic Acid Degradation and the Lethal Effect by Myxin in Escherichia coli

Exposure of Escherichia coli 15T(-) cells to the antibiotic myxin results in the inhibition of deoxyribonucleic acid (DNA) biosynthesis, degradation of intracellular DNA, and death of the cells. Each of these effects was markedly enhanced when protein synthesis was simultaneously inhibited by chloramphenicol. In the continued presence of chloramphenicol, a brief (1 min) exposure to myxin resulted in a rate of DNA degradation and cell death equivalent to that found in the continued presence of myxin alone. Single-strand breaks were present in the DNA of cells exposed to myxin, but when chloramphenicol was also present the breaks were found much earlier. Degradation of DNA in cells exposed to myxin was found to be distributed randomly in both strands and extended over the genome with no restriction to the vicinity of the replication point. There was no release of DNA from its attachment to the cellular membrane in myxin-exposed cells. The possibility that the chloramphenicol effect is due to the inhibition of repair enzyme synthesis which is stimulated by exposure of the cells to myxin is discussed. These data indicate that the extent of the lethal and metabolic damage to the cells by an exposure to myxin represents the result of competition between damage to and repair of cellular DNA.     

Replication of the nuclear genome in yeast does not require concomitant protein synthesis

Incorporation of ³H-adenine into nuclear DNA in a short pulse in mid-S in a synchronised culture of $\text{Saccharomyces cerevisiae}$ was unaffected by the presence of 100 μg/ml cycloheximide. However, colorimetric DNA analyses showed that entry into S was completely blocked by adding the drug at times earlier than about 10 min before initiation of replication. Cell autoradiography of cultures labelled in various regimes showed that at this time there is a cycloheximide-transition point at which the cell acquires the capacity to both initiate and complete a whole round of replication in the presence of 100 μg/ml of cycloheximide. Thus, all the proteins required for passage through one S period are made in advance of initiation.     

Tracing the tracks of genotoxicity by trivalent and hexavalent chromium in Drosophila melanogaster

Mutagen sensitive strains (mus) in Drosophila are known for their hypersensitivity to mutagens and environmental carcinogens. Accordingly, these mutants were grouped in pre- and post-replication repair pathways. However, studying mutants belonging to one particular repair pathway may not be adequate for examining chemical-induced genotoxicity when other repair pathways may neutralize its effect. To test whether both pre-and post-replication pathways are involved and effect of Cr(III)- and Cr(VI)-induced genotoxicity in absence or presence of others, we used double mutant approach in D. melanogaster. We observed DNA damage as evident by changes in Comet assay DNA migration in cells of larvae of Oregon R(+) and single mutants of pre- (mei-9, mus201 and mus210) and post- (mei-41, mus209 and mus309) replication repair pathways and also in double mutants of different combinations (pre-pre, pre-post and post-post replication repair) exposed to increasing concentrations of Cr(VI) (0.0, 5.0, 10.0 and 20.0 μg/ml) for 48 h. The damage was greater in pre-replication repair mutants after exposure to 5.0 μg/ml Cr(VI), while effects on Oregon R(+) and post replication repair mutants were insignificant. Post-replication repair mutants revealed significant DNA damage after exposure to 20.0 μg/ml Cr(VI). Further, double mutants generated in the above repair categories were examined for DNA damage following Cr(VI) exposure and a comparison of damage was studied between single and double mutants. Combinations of double mutants generated in the pre-pre replication repair pathways showed an indifferent interaction between the two mutants after Cr(VI) exposure while a synergistic interaction was evident in exposed post-post replication repair double mutants. Cr(III) (20.0 μg/ml) exposure to these strains did not induce any significant DNA damage in their cells. The study suggests that both pre- and post-replication pathways are affected in Drosophila by Cr(VI) leading to genotoxicity, which may have consequences for metal-induced carcinogenesis.     

UV-induced repair inEscherichia coli B/r Hcr− thy trp cells: Its dependence on UV damage

Irradiation ofEscherichia coli B/r Hcr^? thy trp cells with a low UV-dose permits a post-replication repair of DNA and decreases the breakdown of DNA after a successive irradiation of cells with high UV doses. The usefulness of a repair function of the protein synthesized after a low irradiation dose increases with the increasing damage of DNA.     

Membrane protein synthesis in Escherichia coli: sensitivity to chloramphenicol

Analysis of the labeled proteins of Escherichia coli synthesized in the presence of chloramphenicol (30 or 100 μg/ml) showed: (A) no significant difference in the relative inhibitions of the envelope and cytoplasmic fractions but striking differences in the gel electrophoresis patterns of preparations from chloramphenicol-treated cells versus exponential phase cells; and (B) the average molecular weight of proteins labeled in chloramphenicol (100 μg/ml) was almost half the average of proteins from cells labeled in the absence of chloramphenicol, suggesting that they probably include some incomplete peptides. These results suggest that septation occurs in bacteria by assembly of preexisting envelope proteins in mutants that divide in the presence of high concentrations of chloramphenicol (100 μg/ml).     

Mechanism of replication of phichi174 single-stranded DNA. IV. The parental viral strand is not conserved in the replicating DNA structure

The role of the infecting viral strand in the replication of bacteriophage φX174 replicative form DNA was studied by [³H]thymidine pulse-labeling Escherichia coli cells infected with ²H¹⁵N density-labeled phage. The products of a round of semi-conservative replicative form replication (in light medium) do not contain the original heavy viral strand by 15 minutes after infection or later in the presence of chloramphenicol. Similar results were obtained at earlier times in the absence of chloramphenicol. We conclude that the parental viral strand need not be conserved in the replicating DNA structure in succeeding rounds of replication.     

Temporal sequence of events during the initiation process in Escherichia coli deoxyribonucleic acid replication: roles of the dnaA and dnaC gene products and ribonucleic acid polymerase.

Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of Escherichia coli exposed to the restrictive temperature for one to two generations were examined for the ability to reinitiate DNA replication after returning to the permissive temperature in the presence of rifampin, chloramphenicol, or nalidixic acid. Reinitiation in the dnaA mutant was inhibited by rifampin but not by chloramphenicol, whereas renitiation was not inhibited by rifampin but not by chloramphenicol, whereas reinitiation was not inhibited in two dnaC mutants by either rifampin or chloramphenicol. To observe the rifampin inhibition, the antibiotic must be added at least 10 min before return to the permissive temperature. The rifampin inhibition of reinitiation was not observed when a rifampin-resistant ribonucleic acid ((RNA) polymerase gene was introduced into the dnaA mutant, demonstrating that RNA polymerase synthesizes one or more RNA species required for the initation of DNA replication (origin-RNA). Reinitiation at 30 degrees C was not inhibited by streptolydigin in a stretolydigin-sensitive dnaA muntant. Incubation in the presence of nalidixic acid prevented subsequent reinitiation in the dnaC28 mutant but did not inhibit reinitiation in the dnaA5 muntant. These results demonstrate that the dnaA gene product acts before or during the synthesis of an origin-RNA, RNA polymerase synthesizes this origin RNA, and the dnaC gene product is involved in a step after this RNA synthesis event. Furthermore, these results suggest that the dnaC gene product is involved in the first deoxyribounucleotide polymerization event wheareas the dnaA gene product acts prior to this event. A model is presented describing the temporal sequence of events that occur during initiation of a round of DNA replication, based on results in this and the accompanying paper.     

Characterization of a temperature-sensitive DNA replication mutant of Staphylococcus aureus

A temperature-sensitive DNA replication mutant of Staphylococcus aureus NCTC 8325 has been isolated and characterized. After transfer to the non-permissive-temperature (42 degrees C), DNA synthesis continued for 30 min and the mean DNA content increased by 56%. The amount of residual DNA synthesis was not reduced when the non-permissive temperature was raised, nor when chloramphenicol was added at the time of the temperature shift. During incubation at 42 degrees C, mutant bacteria accumulated the capacity to synthesize DNA after return to the permissive temperature (30 degrees C) in the presence of chloramphenicol. This capacity was lost when chloramphenicol was present at 42 degrees C. The properties of the mutant are consistent with a defect in the initiation of DNA replication at 42 degrees C.     

A novel Escherichia coli mutant capable of DNA replication in the absence of protein synthesis.

An Escherichia coli mutant capable of continued DNA synthesis in the presence of chloramphenicol has been isolated by an autoradiographic technique. The DNA synthesis represents semiconservative replication of E. coli DNA. It can occur in the presence of chloramphenicol or in the absence of essential amino acids, but not in the presence of an RNA synthesis inhibitor, rifampin. The mutant, termed constitutive stable DNA replication (Sdr^c) mutant, appears to grow normally at 37 °C with a slightly slower growth rate than that of the parental strain. DNA replication in the mutant occurs at a reduced rate after 60 minutes in the absence of protein synthesis and continues linearly for several hours thereafter. This distinct slowdown in the DNA replication rate is due to a reduced rate of DNA synthesis in all the cells in the population. Constitutive stable DNA replication appears to require the dnaA and dnaC gene products. The sdr^c mutation has been mapped near the pro-lac region of the E. coli chromosome. The mutation is recessive. Autoradiographic experiments have ruled out the possibility of multiple initiations during a cell cycle. The implication of the above findings is discussed in terms of the regulation of chromosome replication in E. coli.     

Replication of ColE2 and ColE3 plasmids In vitro replication dependent on plasmid-coded proteins

Summary We developed an in vitro replication system for ColE2 and ColE3 plasmids using cell extracts prepared from bacteria with or without these plasmids. DNA synthesis depended on host DNA polymerase I and was sensitive to rifampicin and chloramphenicol. Preincubation of the extracts with plasmid DNA, however, allowed replication of template DNA added subsequently in a plasmid-specific manner in the presence of rifampicin and chloramphenicol. The plasmid-specified trans-acting factor(s) was detected in cell extracts from bacteria carrying a recombinant plasmid with the region of ColE2 or ColE3 encoding the Rep protein. The plasmid-specified factor(s) consisted at least in part of protein, probably the Rep protein. In vitro replication started within a region of ColE2 or ColE3 containing the smallest cis-acting segment essential for in vivo replication and proceeded in a fixed direction.