Induction and repair of double- and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli

Induction and repair of double- and single-strand DNA breaks have been measured after decays of 125I and 3H incorporated into the DNA and after external irradiation with 4 MeV electrons. For the decay experiments, cells of wild type Escherichia coli K-12 were superinfected with bacteriophage lambda DNA labelled with 5'-(125I)iodo-2'-deoxyuridine or with (methyl-3H)thymidine and frozen in liquid nitrogen. Aliquots were thawed at intervals and lysed at neutral pH, and the phage DNA was assayed for double- and single-strand breakage by neutral sucrose gradient centrifugation. The gradients used allowed measurements of both kinds of breaks in the same gradient. Decays of 125I induced 0.39 single-strand breaks per double-strand break. No repair of either break type could be detected. Each 3H disintegration caused 0.20 single-strand breaks and very few double-strand breaks. The single-strand breaks were rapidly rejoined after the cells were thawed. For irradiation with 4 MeV electrons, cells of wild type E. coli K-12 were superinfected with phage lambda and suspended in growth medium. Irradiation induced 42 single-strand breaks per double-strand break. The rates of break induction were 6.75 x 10(-14) (double-strand breaks) and 2.82 x 10(-12) (single-strand breaks) per rad and per dalton. The single-strand breaks were rapidly repaired upon incubation whereas the double-strand breaks seemed to remain unrepaired. It is concluded that double-strand breaks in superinfecting bacteriophage lambda DNA are repaired to a very small extent, if at all.     

Site specificity of bleomycin-mediated single-strand scissions and alkali-labile damage in duplex DNA.

Form II PM2 DNA, which contained bleomycin-mediated single-strand breaks, was purified and treated with the extracellular endonuclease from Alteromonas BAL 31. This enzyme cleaves the phosphodiester backbone opposite a single-strand break to yield a double-strand break. The locations of these double-strand breaks were determined relative to the cleavage sites produced by the restriction enzyme HindIII. The experimental procedure was as follows. Form I PM2 DNA was treated with bleomycin to produce alkali-labile bonds. These were hydrolyzed by alkali treatment and the DNA, now containing single-strand breaks, was purified and treated with the BAL 31 enzyme and the HindIII enzyme to determine the positions of the original alkali-labile bonds. It was found that the single-strand breaks and alkali-labile bonds were introduced at preferred sites on the PM2 genome, since electrophoretic analyses of the DNA after the HindIII digestion revealed DNA bands of discrete sizes. The molecular weights of the DNA fragments produced by these treatments indicate that single-strand breaks and alkali-labile bonds occur at the same sites as those previously determined for direct double-strand scissions introduced by bleomycin at neutral pH. Some of the specific sites of double-strand scissions mediated by bleomycin at neutral pH (Lloyd et al., 1978b) are also shown here to be relatively more reactive than other sites when the DNA contains superhelical turns.     

Radiolysis of chromatin extracted from cultured mammalian cells: production of alkali-labile strand damage in DNA.

Chromatin has been isolated from cultured Chinese-hamster lung fibroblasts as an expanded aqueous gel. The DNA in isolated chromatin has been examined by sedimentation on alkaline sucrose gradients. The average molecular weight of the DNA has been determined to be 50 million. gamma-irradiation of isolated chromatin degrades the DNA to lower molecular weight. The yield of single-strand breaks in the DNA is 0.02 single-strand breaks per krad-10(6) dalton, calculated from a dose-range of &--400 krad and covering a DNA molecular weight range of 2 X 10(7)-1.4 X 10(5). There is a considerable difference in the efficiency of the formation of single-strand breaks in DNA irradiated as isolated chromatin compared with chromatin irradiated in whole cells before isolation. For isolated chromatin, values of 6 dV per break have been calculated compared with about 80 eV per break for chromatin irradiated in whole cells, which suggest a large contribution from indirect action by aqueous radicals in isolated chromatin.     

Escherichia coli radC is deficient in the recA-dependent repair of X-ray-induced DNA strand breaks

Escherichia coli K-12 cells incubated in buffer can repair most of their X-ray-induced DNA single-strand breaks, but additional single-strand breaks are repaired when the cells are incubated in growth medium. While the radC102 mutant was proficient at repairing DNA single-strand breaks in buffer (polA-dependent repair), it was partially deficient in repairing the additional single-strand breaks (or alkali-labile lesions) that the wild-type strain can repair in growth medium (recA-dependent repair), and this repair deficiency correlated with the X-ray survival deficiency of the radC strain. In studies using neutral sucrose gradients, the radC strain consistently showed a small deficiency in rejoining X-ray-induced DNA double-strand breaks, and it was deficient in restoring the normal sedimentation characteristics of the repaired DNA.     

Characterization of intracellular DNA strand breaks induced by neocarzinostatin in Escherichia coli cells.

DNA strand breaks induced by Neocarzinostatin in Escherichia coli cells have been characterized. Radioactively labeled phage lambda DNA was introduced into lysogenic host bacteria allowing the phage DNA to circularize into superhelical molecules. After drug treatment DNA single- and double-strand breaks were measured independently after neutral sucrose gradient sedimentation. The presence of alkali-labile lesions was measured in parallel in alkaline sucrose gradients. The cell envelope provided an efficient protection towards the drug, since no strand breaks were detected unless the cells were made permeable with toluene or with hypotonic Tris buffer. In permeable cells, no double strand breaks could be detected, even at high NCS concentration (100 micrograms/ml). Induction of single-strand breaks leveled off after 15 min at 20 degrees C in the presence of 2 mM mercaptoethanol. Exposure to 0.3N NaOH doubled the number of strand breaks. No enzymatic repair of the breaks could be observed.     

Azaserine: further evidence for DNA damage in Escherichia coli

Azaserine causes DNA damage in stationary-phase cells. In our investigation of this damage, we used strains of Escherichia coli differing in repair capabilities to study azaserine-induced DNA damage, detected as DNA strand breaks by sucrose gradient sedimentation techniques. Reduced sedimentation in alkaline and neutral sucrose gradients indicated the presence of both alkali-labile sites and in situ strand breaks. Azaserine induced DNA single-strand breaks (SSBs) abundantly in all but the recA strain, in which SSBs were greatly reduced. Treatment of purified DNA with azaserine from bacteriophages T4 and PM2 produced no detectable SSBs. Several other studies also failed to detect DNA damage induced directly by azaserine. Increased levels of beta-galactosidase were induced in an E. coli strain possessing a rec::lac fusion, providing further evidence for azaserine induction of the recA gene product. In addition, azaserine induced adaptation against killing but not against mutagenesis in wild-type E. coli strain.     

Alkali-labile sites and post-irradiation effects in single-stranded DNA induced by H radicals.

Single-stranded phiX174 DNA in aqueous solutions has been irradiated in the absence of oxygen, under conditions in which only H radicals react with the DNA. It was shown that H radical reactions result in breaks, which contribute approximately 10 per cent inactivation. Further, two types of alkali-labile sites are formed. One is lethal and gives rise to single-strand breaks by alkali and is most probably identical with post-irradiation heat damage and contributes about 33 per cent to the inactivation mentioned above. The other consists of non-lethal damage, partly dihydropyrimidine derivatives, and is converted to lethal damage by alkali. This follows from experiments in which the DNA was treated with osmium-tetroxide, which oxidizes thymine to 5,6-dihydroxy-dihydrothymine. Treatment with alkali of this DNA gives the same temperature dependence as found for the non-lethal alkali-labile sites in irradiated DNA. A similar temperature dependence is found for dihydrothymine and irradiated pyrimidines with alkali.     

Absence of swiveling at sites of intercalator-induced protein-associated deoxyribonucleic acid strand breaks in mammalian cell nucleoids

The sedimentation of DNA-nuclear protein complexes in 1.9 M salt-neutral sucrose gradients (nucleoid sedimentation) was used to examine the effects of the DNA intercalator 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) on mouse leukemia cell DNA. Mild detergent cell lysis and neutral pH make nucleoid sedimentation an extremely gentle, but sensitive, method to detect DNA scission. DNA breaks reduce the compaction of nucleoids and slow their sedimentation. Nucleoids from m-AMSA-treated cells sedimented as did those from untreated cells, indicating no detectable m-AMSA-dependent alterations in compaction despite an apparent underlying DNA break frequency of approximately 3 per 10(6) nucleotides, as measured by alkaline elution with proteinase. Mild proteinase digestion of cell lysates prior to nucleoid sedimentation unmasked some, but not all, of the underlying breaks. The frequency of DNA-protein cross-links in nucleoids from cells treated with m-AMSA was comparable to the single-strand break frequency produced by m-AMSA in whole cells. These results indicate that m-AMSA-induced DNA-protein cross-links conceal DNA breaks so as to prevent swiveling around the breaks within the nucleoids. This unique sort of DNA scission is consistent with the involvement of topoisomerases in the DNA breaks elicited by intercalators in mammalian cells.     

Ionizing radiation damage to the folded chromosome of Escherichia coli K-12: sedimentation properties of irradiated nucleoids and chromosomal deoxyribonucleic acid.

The structures of the membrane-free nucleoid of Escherichia coli K-12 and of unfolded chromosomal deoxyribonucleic acid (DNA) were investigated by low-speed sedimentation on neutral sucrose gradients after irradiation with 60Co gamma rays. Irradiation both in vivo and in vitro was used as a molecular probe of the constraints on DNA packaging in the bacterial chromosome. The number of domains of supercoiling was estimated to be approximately 180 per genome equivalent of DNA, based on measurements of relaxation caused by single-strand break formation in folded chromosomes gamma irradiated in vivo and in vitro. Similar estimates based on the target size of ribonucleic acid molecules responsible for maintaining the compact packaging of the nucleoid predicted negligible unfolding due to the formation of ribonucleic acid single-strand breaks at doses of up to 10 krad; this was born out by experimental measurements. Unfolding of the nucleoid in vitro by limit digestion with ribonuclease or by heating at 70 degrees C resulted in DNA complexes with sedimentation coefficients of 1,030 +/- 59S and 625 +/- 15S, respectively. The difference in these rates was apparently due to more complete deproteinization and thus less mass in the heated material. These structures are believed to represent intact, replicating genomes in the form of complex-theta structures containing two to three genome equivalents of DNA. The rate of formation of double-strand breaks was determined from molecular weight measurements of thermally unfolded chromosomal DNA gamma irradiated in vitro. Break formation was linear with doses up to 10 krad and occurred at a rate of 0.27 double-strand break per krad per genome equivalent of DNA (1,080 eV/double-strand break). The influence of possible nonlinear DNA conformations on these values is discussed.     

Growth-medium-dependent repair of DNA single-strand and double-strand breaks in X-irradiated Escherichia coli

The X-ray resistance of logarithmic phase cells of Escherichia coli K-12 is enhanced threefold by growth in rich medium versus minimal medium (N. J. Sargentini, W. P. Diver, and K. C. Smith, Radiat. Res. 93, 364-380, 1983). In this work, X-ray-induced DNA strand breaks were assayed by sedimentation in alkaline and neutral sucrose gradients to correlate the enhanced survival of rich-medium-grown cells with an enhanced capacity for DNA repair. While rich-medium-grown cells showed no enhanced capacity for repairing DNA single-strand breaks in buffer, i.e., fast, polA-dependent repair, they did show an enhanced capacity to repair both single-strand and double-strand breaks in growth medium, i.e., slow, recA-dependent repair. This enhanced capacity for DNA repair in rich-medium-grown cells was inhibited by rifampicin post-treatment, indicating the requirement for de novo RNA synthesis. Kinetic studies indicated that the repair of DNA double-strand breaks was a complex process. Relative to the sedimentation rate in neutral sucrose gradients of nonirradiated DNA, the sedimentation rate of X-irradiated DNA first changed from slow to very fast. Based on alkaline sucrose gradient sedimentation studies, all the strand breaks had been repaired during the formation of the very fast sedimenting DNA. With continued incubation, the sedimentation rate of the DNA on neutral sucrose gradients decreased to the normal rate.     

DNA strand break and rejoining in cultured human fibroblasts exposed to fast neutrons or gamma rays

The production and rejoining of DNA single-strand and double-strand breaks have been monitored in monolayer cultures of proliferating human skin fibroblasts by means of sensitive techniques. Cells were irradiated with low doses of either 60Co gamma-rays or 14.6 MeV neutrons at 0 degrees C (0-5 Gy for measurement of single-strand breaks by alkaline elution and 0-50 Gy for double-strand breaks measured by neutral elution). The yield of single-strand breaks induced by neutrons was 30 per cent of that produced by the same dose of gamma-rays; whilst in the induction of double-strand breaks neutrons were 1.6 times as effective as gamma-rays. Upon post-irradiation incubation of cells at 37 degrees C, neutron-induced single-strand and double-strand breaks were rejoined with a similar time-course to gamma-induced breaks. Rejoining followed biphasic kinetics; of the single-strand breaks, 50 per cent disappeared within 2 min after gamma-rays and 6-10 min after neutrons. Fifty per cent of the double-strand breaks disappeared within 10 min, after gamma-rays and neutrons. Cells derived from patients suffering from ataxia-telangiectasia showed the same capacity for repair of single- and double-strand breaks induced by 14.6 MeV neutrons, as cells established from normal donors. The comparison of neutrons and gamma-rays in the induction of DNA breaks did not explain the elevated r.b.e. on high LET radiation. However, a study of the variation in the spectrum of lesions induced by different radiation sources will probably contribute to the clarification of the relative importance of other radio products.     

Single-strand breakage in DNA of Escherichia coli exposed to Cd2+.

When a growing culture of Escherichia coli was exposed to 3 X 10(-6) M Cd2+, 85 to 95% of the cells lost their ability to form colonies on agar plates. Loss of viability was accompanied by considerable single-strand breakage in the DNA, with no detectable increase in double-strand breaks. A direct correlation appeared to exist between the number of single-strand breaks and the concentrations of Cd2+ to which the cells were exposed. Exposure of DNA in vitro to a Cd2+ concentration of 3 X 10(-6) M or higher, followed by sedimentation in alkaline sucrose gradients, demonstrated no single-strand breaks. Cadmium-exposed cells recovered viability when incubated in Cd2+-free liquid medium containing 10 mM hydroxyurea. During the early period of recovery, there was a lag in the incorporation of labeled thymidine, but cellular DNA, at least in part, appeared to be repaired.     

Damage and Repair of DNA in 5-Bromodeoxyuridine-Labeled Chinese Hamster Cells Exposed to Fluorescent Light

Illumination of Chinese hamster cells with fluorescent light after 5-bromodeoxyuridine incorporation leads to extensive single-strand breakage in the DNA of the exposed cells. The rate of production of single-strand breaks is dependent on the extent to which thymine is replaced by 5-bromouracil. At least some of the breaks observed with alkaline gradients are probably produced in vivo and are probably not contingent upon alkaline hydrolysis since breakage can be demonstrated with neutral gradients also. Cells are able to rejoin most of the single-strand breaks within 60 min; however, damage to the DNA-containing material (the “complex”) initially released from cells is repaired more slowly. Cysteamine protects against single-strand breakage with a dose-modifying factor of 2.8. A comparison is made between the production of single-strand breaks by fluorescent light and X-rays, and the significance of such breaks relative to cell survival is discussed.     

Repair of DNA single-strand breaks in radiation-sensitive mutants of mouse cells

The radiation-sensitive mutant M10 of mouse lymphoma L5178Y cells was examined for its ability to rejoin DNA single-strand breaks induced by gamma-rays. The alkaline sucrose gradient sedimentation analysis revealed that M10 cells repaired single-strand breaks but simultaneously produced increasing amounts of small DNA fragments with time of postirradiation incubation, something which was not observed in L5178Y cells. Since small fragments did not appear in M10 cells irradiated at room temperature, DNA fragmentation may result from cold treatment during irradiation followed by incubation at 37 degrees C. This indicates that the cold susceptibility is characteristic of M10 cells and is not related to radiation sensitivity of this mutant. This conclusion is supported by the finding that no DNA degradation takes place after cold treatment with a subsequent incubation in the other radiosensitive mutant LX830 that belongs to the same complementation group as M10.     

Neutral sucrose gradient sedimentation of very large DNA from Bacillus subtilis. II. Double-strand breaks formed by gamma ray irradiation of the cells

A procedure has been developed whereby essentially all the DNA from Bacillus subtilis cells can be reproducibly extracted in a form which sediments 2.3 times faster than bacteriophage T2 DNA in a neutral sucrose gradient spun at 20,000 revs/ min. When the cells are irradiated with low (3 to 34 kilorads) gamma ray doses, some DNA moves in a slower peak, which from the previous paper (Levin &; Hutchinson, 1973) appears to be linear DNA. Some of the DNA also sediments ahead of the unirradiated DNA. On incubation of the cells at 37°C under conditions such that single-strand DNA breaks are repaired, the fast-sedimenting component is partially restored, with the DNA sedimenting ahead of it usually disappearing, and the quantity in the slower component decreasing. With 80 minutes incubation the fraction of DNA after various radiation doses in the fast-sedimenting component is the same as the fraction of cells able to form colonies, suggesting that the destruction of the component is responsible for the effect of gamma rays on the ability of a cell to replicate. Single-strand breaks introduced into the DNA within the cells do not affect the fast-sedimenting component, so radiation-induced single-strand breaks are not responsible for the effect of gamma rays on replication.The double-strand break rate for DNA in the cells is 0.010 breaks per mass the size of T2 DNA per kilorad. The fast-sedimenting component in irradiated cells which have not been incubated disappears at a rate equal to one radiationinduced double-strand break formed per genome. Since the fast-sedimenting component in solution is also destroyed by one double-strand break per genome (Levin &; Hutchinson, 1973), it is suggested that this component is the genome in the form of a circle. The correspondence between DNA in the fast-sedimenting form after incubation and the ability of cells to form colonies then indicates that a genome can replicate only if all double-strand breaks are repaired.     

Repair of DNA double-strand breaks in Escherichia coli, which requires recA function and the presence of a duplicate genome.

A method was devised for extracting, from cells of Escherichia coli K12, DNA molecules which sedimented on neutral sucrose gradients as would be expected for free DNA molecules approaching the genome in size. Gamma ray irradiation of oxygenated cells produced 0.20 DNA double-strand breaks per kilorad per 10⁹ daltons. Incubation after irradiation of cells grown in K medium, with four to five genomes per cell, showed repair of the double-strand breaks. No repair of double-strand breaks was found in cells grown in aspartate medium, with only 1.3 genomes per cell, although DNA single-strand breaks were still efficiently repaired. Cells which were recA^? or recA^?recB^? also did not repair double-strand breaks. These results suggest that repair of DNA double-strand breaks may occur by a recombinational event involving another DNA double helix with the same base sequence.     

DBD-fish on neutral comets: simultaneous analysis of DNA single- and double-strand breaks in individual cells

Humanblood leukocytes exposed to X-rays were immersed in an agarose microgel on a slide, extensively deproteinized, and electrophoresed under neutral conditions. Following this single-cell gel electrophoresis assay, characteristics of DNA migration (i.e., area of the comet) are related to the DNA double-strand breaks (dsbs) yield. After electrophoresis, comets were briefly incubated in an alkaline unwinding solution, transforming DNA breaks and alkali-labile sites into restricted single-stranded DNA (ssDNA) motifs. These motifs behave as target sites for hybridization with a whole genome probe, following the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure. As DNA breakage increases with dose, more ssDNA is produced in the comet by the alkali and more DNA probe hybridizes, resulting in an increase in the mean fluorescence intensity. Since radiation-induced DNA single-strand breaks (ssbs) are far more frequent than dsbs, the mean fluorescence intensity of the DBD-FISH signal from the comet is related to the ssb level, whereas the surface area of the same comet signal is indicative of the dsb yield. Thus, both DNA break types may be simultaneously analyzed in the same cell. This was confirmed in a repair assay performing the DBD-FISH on neutral comets from a human cell line defective in the repair of dsbs. Otherwise, treatment with hydrogen peroxide, a main inducer of ssbs, increased the mean fluorescence intensity, but not the surface, of X-ray-exposed human leukocytes.     

The effect of pentachlorophenol and its metabolite tetrachlorohydroquinone on cell growth and the induction of DNA damage in Chinese hamster ovary cells

It is shown that p-tetrachlorohydroquinone (TCH), the metabolite of the environmental chemical pentachlorophenol (PCP), is more toxic to cultured CHO cells than PCP, and that it causes DNA single-strand breaks and/or alkali-labile sites at concentrations of 2-10 microgram/ml as demonstrated by the alkaline elution technique.     

Relative rates of repair of single-strand breaks and postirradiation DNA degradation in normal and induced cells of Escherichia coli.

Labeled DNA from irradiated Excherichia coli cells has been studied on an alkaline sucrose gradient without acid precipitation of the DNA. This enables the observation of both DNA repair and DNA degradation. The use of a predose of ultraviolet light (UV) causes induction of an inhibitor of postirradiation DNA degradation in lex+ strains. The effect of this induction on both the repair of single-strand breaks and DNA degradation has been followed in strains WU3610 (uvr+) and WU3610-89 (uvr-). The repair process is more rapid than the degradation, and when degradation is inhibited more repair is apparent. Cells that are lex- (Bs-1 and AB2474) cannot be induced for inhibition of degradation. Nevertheless, by observation at short times repair can be seen clearly. This repaired DNA is degraded, suggesting that the signal for DNA degradation is not a single-strand break.     

Alkali lability and rapid initiation of excision repair following photoaffinity damage by ethidium azide

DNA damage and repair provoked by ethidium azide (EA) photoaffinity labeling in mouse leukemia cells was studied by measuring sedimentation properties of nucleoids in neutral sucrose gradients, and it was found that the strand opening step was faster than that which followed damage of cells by ultraviolet (UV) light. The two insults were compared at levels of damage which gave the same overall rates of repair synthesis in intact cells and which required the same length of time to complete repair, as judged by the restoration of supercoiling of the isolated nucleoids. In the case of UV, single-strand breaks in DNA were detectable at 30 min, maximum at 2 h, and the superhelical properties restored at 21 h. With photoaffinity labeling, single-strand breaks were prominent immediately, even when photolabeling of cells was done on ice, but restoration of DNA supercoiling still required 21 h. Photolabeling of isolated nucleoids or isolated viral DNA with EA failed to introduce DNA strand breaks. However, it was discovered that photoaffinity labeling of DNA with EA resulted in alkali labile sites shown by single strand breaks produced on alkaline sucrose sedimentation or by alkali exposure followed by sedimentation on neutral formamide gradients. These results suggest that the drug attachment sites should be identifiable by the location of such single strand breaks.