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.     

DNA damage and biological expression of adenovirus: a comparison of liquid versus frozen conditions of exposure to gamma rays

Human adenovirus type 2 (Ad 2) was irradiated with 137Cs gamma rays in the liquid state at 0 degree C. DNA breaks were correlated with the inactivation of several viral functions and compared to results obtained previously for irradiation of Ad 2 under frozen conditions at -75 degrees C. Irradiation at 0 degree C induced 170 +/- 20 single-strand breaks and 2.6 +/- 0.4 double-strand breaks/Gy/10(12) Da in the viral DNA. Viral adsorption to human KB cells was inactivated with a D0 of 9.72 +/- 1.18 kGy, whereas the inactivation of Ad 2 plaque formation had a D0 of 0.99 +/- 0.14 or 1.1 +/- 0.29 kGy when corrected for the effect of radiation on virus adsorption. For the adsorbed virus, an average of 4.3 +/- 1.7 single-strand and 0.065 +/- 0.02 double-strand breaks were induced in the viral DNA per lethal hit. In contrast, irradiation of Ad 2 at -75 degrees C results in 2.6- to 3.4-fold less DNA breakage per Gy and a 5.6-fold increase in D0 for plaque formation of the adsorbed virus. Furthermore, although host cell reactivation (HCR) of Ad 2 viral structural antigen production for irradiated virus was substantially reduced in the xeroderma pigmentosum fibroblast strain (XP25RO) compared to normal strains for irradiation at -75 degrees C (57% HCR), it was only slightly reduced compared to normal for irradiation at 0 degree C (88% HCR). These results indicate that the spectrum of DNA damage is both quantitatively and qualitatively different for the two conditions of irradiation.     

DNA damage and repair in relation to cell killing in neocarzinostatin-treated HeLa cells.

To elucidate the mechanism of the cell killing activity of neocarzinostatin on mammalian cells, the drug-induced damage of DNA and its repair were examined. Very low doses of neocarzinostatin, at which high survival of cells was observed, clearly produced single-strand breaks of DNA and decomposition of the 'DNA complex', but these damages appeared to be repaired almost completely. At higher doses of neocarzinostatin, single-strand breaks were repaired to a considerable extent while double-strand breaks seemed not to be repaired. The number of non-repairable single-strand breaks was about twice that of double-strand breaks. This implies that single-strand breaks are repaired except for those constituting double-strand breaks. Although at low levels of neocarzinostatin repair of double-strand breaks may occur, the correlation existing between the colony-forming ability of cells treated with neocarzinostatin and non-repairable DNA breakage suggests that production of a small number of critical non-repairable double-strand breaks per cell may be responsible for the cell killing activity of the drug.     

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.     

Gamma-ray induced double-strand breaks in DNA resulting from randomly-inflicted single-strand breaks: temporal local denaturation, a new radiation phenomenon?

The induction of single- and double-strand breaks in DNA by gamma-rays has been measured. The maximum number of nucleotide pairs (a) between two independently induced single-strand breaks in opposite strands of the DNA which cannot prevent the occurrence of a double-strand break was found to amount to about 16. This value did not differ significantly for the four types of bacteriophage DNA investigated (T4, T7 and PM2 DNA, and replicative form DNA of phage phiX174) and was the same in 10(-2) M phosphate buffer containing 0, 0.5 or 1 M NaCl. In 10(-3) M phosphate buffer a was 34 nucleotide pairs. Evidence is presented that the relatively large value of a has to be ascribed at least partly to a temporal local denaturation accompanying the induction of a single-strand scission. A contribution of base damage that labilizes the DNA-helix, between two single-strand breaks to the high value of a can not be excluded.     

Mammalian DNA single-strand break repair: an X-ra(y)ted affair

The genetic stability of living cells is continuously threatened by the presence of endogenous reactive oxygen species and other genotoxic molecules. Of particular threat are the thousands of DNA single-strand breaks that arise in each cell, each day, both directly from disintegration of damaged sugars and indirectly from the excision repair of damaged bases. If un-repaired, single-strand breaks can be converted into double-strand breaks during DNA replication, potentially resulting in chromosomal rearrangement and genetic deletion. Consequently, cells have adopted multiple pathways to ensure the rapid and efficient removal of single-strand breaks. A general feature of these pathways appears to be the extensive employment of protein-protein interactions to stimulate both the individual component steps and the overall repair reaction. Our current understanding of DNA single-strand break repair is discussed, and testable models for the architectural coordination of this important process are presented.     

Contributions of various types of damage to inactivation of T4 bacteriophage by protons

Mean doses for damage induced by 3.7-MeV protons in T4 phage were measured for the following effects: inactivation, killing, adsorption, DNA injection, capsid rupture with DNA release, and single- and double-strand DNA breaks. These effects have been related to phage survival in the same experiment because of the variability inherent in such measurements. The experiments were carried out in nutrient broth, phosphate buffer, and phosphate buffer plus histidine as suspension media. The following conclusions can be drawn: (i) DNA double-strand breakage is the dominant cause of inactivation in nutrient broth; (ii) scavengers protect the DNA inside the capsid to only a small degree; (iii) indirect actions affect functions associated with proteins; (iv) DNA release, as measured by capsid rupture, accounts for only a small percentage of the loss of viability; (v) essentially all DNA from adsorbed phage is injected even though a large proportion of the DNA contains double-strand breaks.     

Radiation-Induced Breaks of DNA in Cultured Mammalian Cells

Mouse leukemic cells (L5178Y) in suspension culture were irradiated and the extent of single-strand breaks and double-strand cuts of DNA was estimated by sucrose gradient centrifugation. The radiation produced 3.0 single-strand breaks per cell (G(1) stage) per rad and approximately 0.3 double-strand breaks per cell (G(1) stage) per rad.     

Single- and double-strand breaks in 5-bromouracil-substituted DNA of B. subtilis and phage PBSH after irradiation with long-wave length UV and their correlation to intramolecular energy transfer

5-Bromouracil-substituted DNA was isolated form B. Subtilis and phage PBSH. The three DNA fractions of Different densities (TT, TB, and BB) were irradiated with u.v. (313nm). The number of single-strand and double-strand breaks was determined. The breakage rates are given. It was found that in hybrid DNA (TB) double-strand breaks occur depending linearly on dose. In BB–DNA the observed double-Strand bresks can be divided into two fractions with a linear and quadratic dose dependence respectively. The results can be explained by assuming intramolecular energy transfer from the BU-containing strand to its complementary strand.     

Rejoining kinetics of DNA single- and double-strand breaks in normal and DNA ligase-deficient cells after exposure to ultraviolet C and gamma radiation: an evaluation of ligating activities involved in different DNA repair processes

The repair kinetics for rejoining of DNA single- and double-strand breaks after exposure to UVC or gamma radiation was measured in cells with deficiencies in DNA ligase activities and in their normal counterparts. Human 46BR cells were deficient in DNA ligase I. Hamster EM9 and EM-C11 cells were deficient in DNA ligase III activity as a consequence of mutations in the XRCC1 gene. Hamster XR-1 cells had mutation in the XRCC4 gene, whose product stimulates DNA ligase IV activity. DNA single- and double-strand breaks were assessed by the comet assay in alkaline conditions and by the technique of graded-field gel electrophoresis in neutral conditions, respectively. 46BR cells, which are known to re-ligate at a reduced rate the DNA single-strand breaks incurred during processing of damage induced by UVC but not gamma radiation, were shown to have a normal repair of radiation-induced DNA double-strand breaks. EM9 cells exhibited a reduced rate of rejoining of DNA single-strand breaks after exposure to ionizing radiation, as reported previously, as well as UVC radiation. EM-C11 cells were deficient in the repair of radiation-induced-DNA single-strand breaks but, in contrast to EM9 cells, demonstrated the same kinetics as the parental cell line in the resealing of DNA breaks resulting from exposure to UVC radiation. Both EM9 and EM-C11 cells displayed a significant defect in rejoining of radiation-induced-DNA double-strand breaks. XR-1 cells were confirmed to be highly deficient in the repair of radiation-induced DNA double-strand breaks but appeared to rejoin DNA single-strand breaks after UVC and gamma irradiation at rates close to normal. Taken together these results indicate that: (1) DNA ligase I is involved only in nucleotide excision repair; (2) DNA ligase IV plays an important role only in repair of DNA double-strand breaks; and (3) DNA ligase III is implicated in base excision repair and in repair of DNA double-strand breaks, but probably not in nucleotide excision repair.     

Exponential or shouldered survival curves result from repair of DNA double-strand breaks depending on postirradiation conditions

The yeast mutant rad54-3 is temperature conditional for the rejoining of DNA double-strand breaks, but cells do proliferate at both the restrictive and permissive temperatures. Thus, after irradiation with 30 MeV electrons, survival curves can be obtained which may or may not involve double-strand break rejoining under certain experimental conditions. Because of this special property of rad54-3 cells, it was possible to demonstrate that rejoining of radiation-induced double-strand breaks under nongrowth conditions yields exponential survival curves the slopes of which decrease as a function of the rejoining time. These survival data suggest that, under nongrowth conditions, the rejoining of double-strand breaks is an unsaturated process and lacks binary misrepair. In contrast, whenever rejoining of double-strand breaks occurs under growth conditions, shouldered survival curves are observed. This is true for immediate plating as well as for delayed plating survival curves. It is proposed that it is the unsaturated rejoining of double-strand breaks under nongrowth conditions, lacking binary misrepair, which is responsible for potentially lethal damage repair.     

Radiation-induced strand breaks in phi X174 replicative form DNA: an improved experimental and theoretical approach

To determine the yield of radiation-induced single-strand, double-strand and potential breaks (breaks which are converted into actual breaks by alkali or heat treatment) oxygenated aqueous solutions of phi X174 supercoiled circular double-stranded (RFI) DNA were irradiated with increasing doses of gamma-irradiation and subjected to electrophoresis on agarose gels both before and after heat treatment. A complete separation was obtained of RFI, RFII (relaxed circle due to one or more single-strand breaks) and RFIII (linear DNA due to one double-strand break). A computer-assisted spectrophotometric procedure was developed, which enabled us to measure very accurately the amount of DNA present in the three DNA fractions. The quantitative changes of each fraction of DNA with dose could be fitted to a straightforward statistical model, which described the dose-dependent formation of the different types of breaks and from which the D37-values of single-strand, potential single-strand and double-strand breaks could be calculated to be 0.42 +/- 0.02, 1.40 +/- 0.25 and 57 +/- 36 Gy respectively. Potential double-strand breaks were not formed significantly under our conditions. In addition the maximum distance between two independently introduced single-strand breaks in opposite strands resulting in a double-strand break could be determined. The values before and after heat treatment are shown to be 29 +/- 6 and 102 +/- 13 nucleotides, respectively.     

Single-strand breakage on binding of DNA to cells in the genetic transformation of Diplococcus pneumoniae.

Mutants of Diplococcus pneumoniae that lack a membrane-localized DNAase are defective in transformation because entry of DNA into the cell is blocked. Such mutants still bind DNA on the outside of the cell. The bound DNA is double-stranded and its double-stranded molecular weight is unchanged. Its sedimentation behavior in alkali, however, shows that it has undergone single-strand breakage. The breaks are located randomly in both strands of the bound DNA at a mean separation of 2 × 10⁶ daltons of single-stranded DNA. Both binding and single-strand breakage occur in the presence of EDTA. Single-strand breaks are similarly formed on binding of DNA to normally transformable cells in the presence of EDTA. The single-strand breaks appear to be a consequence of attachment. DNA may be bound to the cell surface at the point of breakage.A mutant that is partially blocked in entry also binds DNA mainly on the outside of the cell. In the presence of EDTA, DNA bound by this mutant undergoes only single-strand breaks. In the absence of EDTA, however, double-strand breaks occur, apparently as a result of the initiation of entry. It is possible that the double-strand breaks arise from additional single-strand breaks opposite those that occurred on binding. The double-strand breaks presumably result from action of the membrane DNAase as it begins to release oligonucleotides from one strand segment while drawing the complementary strand segment into the cell.     

Effects of x irradiation on a temperate bacteriophage of Haemophilus influenzae.

The inactivation of bacteriophage HP1c1 by X rays in a complex medium was found to be exponential, with a D0 (the X-ray exposure necessary to reduce the survival of the phage to 37%) of approximately 90 kR. Analysis of results of sucrose sedimentation of DNA from X-irradiated whole phage showed that the D0 for intactness of single strands was about 105kR, and for intactness of double strands, it was much higher. The D0 for attachment of X-irradiated phage to the host was roughly estimated as about 1,100 kR. Loss of DNA from the phage occurred and was probably due to lysis of the phage by X irradiation, but the significance of the damage is not clear. The production of single-strand breaks approaches the rate of survival loss after X irradiation. However, single-strand breaks produced by UV irradiation, in the presence of H2O2, equivalent to 215 kR of X rays, showed no lethal effect on the phage. Although UV-sensitive mutants of the host cell, Haemophilus influenzae, have been shown to reactivate UV-irradiated phage less than does the wild-type host cell, X-irradiated phage survive equally well on the mutants as on the wild type, a fact suggesting that other repair systems are involved in X-ray repair.     

Characterization and quantitation of DNA strand breaks requiring recA-dependent repair in X-irradiated Escherichia coli

The repair of X-ray-induced DNA single-strand breaks was studied after the completion of growth-medium-independent repair in Escherichia coli K-12. A comparison of the sedimentation of DNA from bacteriophages T2 and T7 was used to test the accuracy of our alkaline and neutral sucrose gradient procedures for determining the molecular weight of bacterial DNA. The repair of DNA single-strand breaks by cells incubated in buffer occurred by two processes. About 85% of the repairable breaks were resealed rapidly (t1/2 = less than 6 min), while the remainder were resealed slowly (t1/2 = approximately 20 min). After the completion of the repair of DNA single-strand breaks in buffer, about 80% of the single-strand breaks that remained were found to be associated with DNA double-strand breaks. The subsequent resuspension of cells in growth medium allowed the repair of both DNA single- and double-strand breaks in wild-type but not in recA cells. Thus the recA-dependent, growth-medium-dependent repair of DNA single-strand breaks is essentially the repair of DNA double-strand breaks.     

Induction of chromosome damage by Neurospora endonuclease in repair-inhibited quiescent normal human fibroblasts

The purpose of these experiments was to determine the role of double-strand breaks in chromosome aberration formations. Quiescent normal human fibroblasts were treated with 3 μM nitrogen mustard and then allowed to repair their DNA damage for 24 h prior to cell fusion and induction of premature chromosome condensation. The extent of chromosome damage was determined in the G1 prematurely condensed chromosomes (G1 PCC). The presence of cytosine arabinoside and hydroxyurea during the repair period in order to accumulate single-strand DNA breaks resulted in an increase in the chromosome-break frequency. Treatment of these repair-inhibited cells with single-strand-specific neurospora endonuclease during fusion to change single-strand lesions into double-strand breajs resulted in a doubling of the aberration frequency. These results support the notion that double-strand breaks are important in chromosome-aberration formation.     

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.     

Double-strand DNA break formation mediated by flap endonuclease-1

Double-strand DNA breaks are the most lethal type of DNA damage induced by ionizing radiations. Previously, we reported that double-strand DNA breaks can be enzymatically produced from two DNA damages located on opposite DNA strands 18 or 30 base pairs apart in a cell-free double-strand DNA break formation assay (Vispé, S., and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392). In the assay that we developed, these two DNA damages are converted into single-strand interruptions by enzymes involved in base excision repair. We showed that these single-strand interruptions are converted into double-strand DNA breaks; however, it was not due to spontaneous denaturation of DNA. Thus, we proposed a model in which DNA polymerase delta/epsilon, by producing repair patches at single-strand interruptions, collide, resulting in double-strand DNA break formation. We tested the model and investigated whether other enzymes/factors are involved in double-strand DNA break formation. Here we report that, instead of DNA polymerase delta/epsilon, flap endonuclease-1 (FEN-1), an enzyme involved in base excision repair, is responsible for the formation of double-strand DNA break in the assay. Furthermore, by transfecting a flap endonuclease-1 expression construct into cells, thus altering their flap endonuclease-1 content, we found an increased number of double-strand DNA breaks after gamma-ray irradiation of these cells. These results suggest that flap endonuclease-1 acts as a double-strand DNA break formation factor. Because FEN-1 is an essential enzyme that plays its roles in DNA repair and DNA replication, DSBs may be produced in cells as by-products of the activity of FEN-1.     

The accumulation of single-stranded breaks does not lead to paired DNA damage--the characteristic of the transcribing fragment of the human ribosomal operon that allows its being detected in biological fluids at the death of different body cells

It was shown by blot-hybridization with corresponding DNA probes after electrophoretic separation of control and experimental samples of human genome DNA that accumulation of single-strand breaks in the chains of double-strand fragment of transcribing range of ribosomal gene (TRrDNA) does not result in double-strand breaks. That differs from the other studied DNA sequences (cluster of histon genes, Alu-repetition, telomeric repetition and satellite III). Single-strand breaks and double-strand breaks were induced by endonucleases and by gamma-radiation. In spite of higher chemical modification of TRrDNA by arylazide and dimethylsulfate (because of high content of GC-pairs), under the following fragmentation TRrDNA was found to be more resistant to double-strand breaks than other studied DNA sequences. At the same time in the range of non-transcribing spacer (NTS) of ribosomal gene, the section with higher sensitivity to double-strand breaks was found. Higher resistance of TRrDNA to double breaks makes it possible to identify these fragments in cell material from different tissue after death or in DNA samples after prolonged storage. Resistance of TRrDNA to formation of double-strand breaks can be used for its detection in biological fluids after cell death, including the death initiated by ionizing radiation.     

DNA double-strand breaks and alkali-labile bonds produced by bleomycin.

Both in linear T2 DNA, analyyzed by velocity sedimentation, and in supercoiled Col EL DNA, analyzed by gel electrophoresis, the number of double-strand breaks produced by bleomycin was directly propotional to the number of single-strand breaks and was far greater than the number expected from random coincidence of single-strand breaks, suggesting that the bleomycin-induced double-strand breaks occur as an independent event. In Col EL DNA, at least twice as many single-strand breaks were found under alkaline assay conditions as were found under neutral conditions, showing the production of alkaline-labile bonds by bleomycin.