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.     

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.     

Radiation-induced DNA single-strand breaks in freshly isolated human leukocytes.

Single-strand breaks are a major form of DNA damage caused by ionizing radiation, and measurement of strand breaks has long been used as an index of overall cellular DNA damage. Most assays for DNA single-strand breaks in cells rely on measuring fractionated DNA samples following alkali denaturation. Quantification is usually achieved by prelabeling cells with radioactive DNA precursors; however, this is not possible in the situation of nondividing cells or freshly isolated tissue. It has previously been demonstrated that the alkali unwinding assay of DNA strand breaks can be quantified by blotting the recovered DNA on nylon membranes and hybridizing with radiolabeled sequence-specific probes. We report here improvements to the technique, which include hot alkali denaturation of DNA samples prior to blotting and the use of carrier DNA that is non-complementary to the radiolabeled probe. Our method allows both single- and double-stranded DNA to be quantified with the same efficiency, thereby improving the sensitivity and reproducibility of the assay, and allows calibration for determination of absolute levels of DNA strand breaks in cells. We also used this method to assay radiation-induced DNA strand breaks in freshly isolated human leukocytes and found them to have a strand break induction rate of 1815 strand breaks/cell/Gy.     

Postreplicational formation and repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB cells

The number of DNA double-strand breaks formed in UV-irradiated uvrB recF recB cells correlates with the number of unrepaired DNA daughter-strand gaps, and is dependent on DNA synthesis after UV-irradiation. These results are consistent with the model that the DNA double-strand breaks that are produced in UV-irradiated excision-deficient cells occur as the result of breaks in the parental DNA opposite unrepaired DNA daughter-strand gaps. By employing a temperature-sensitive recA200 mutation, we have devised an improved assay for studying the formation and repair of these DNA double-strand breaks. Possible mechanisms for the postreplication repair of DNA double-strand breaks are discussed.     

Inhibition and recovery of DNA synthesis in human tumor cell lines following radiation exposure

Eight human tumor cell lines with radiosensitivities (D0) ranging from 1 to 3 Gy were analyzed for their response to radiation-induced inhibition of DNA synthesis. These cell lines differ in their sensitivity to induction of DNA double-strand breaks and in the rate at which they rejoin DNA double-strand breaks. Fifty-gray doses of gamma rays induced between 35 and 75% inhibition in rates of DNA synthesis. The magnitude of the inhibition was not related to cellular radiosensitivity, frequency of initial DNA double-strand breaks, or the rate of rejoining of DNA double-strand breaks. All the cell lines studied had similar kinetics of recovery from inhibition of DNA synthesis following radiation exposure. These results suggest that factors other than or in addition to frequency of DNA double-strand breaks are important in the control of DNA synthesis following exposure to ionizing radiation in human tumor cell lines.     

Changes of 8-OH-dG levels in DNA and its base excision repair activity in rat lungs after inhalation exposure to hexavalent chromium

The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl-N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl2, the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl-N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl2 the MMS-induced DNA strand breaks accumulated during the first 2h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair is an important mechanism of cadmium induced mutagenicity and carcinogenicity.     

X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution.

This work presents a neutral filter elution method for detecting DNA double strand breaks in mouse L1210 cells after X-ray. The assay will detect the number of double strand breaks induced by as little as 1000 rad of X-ray. The rate of DNA elution through the filters under neutral conditions increases with X-ray dose. Certain conditions for deproteinization, pH, and filter type are shown to increase the assay's sensitivity. Hydrogen peroxide and Bleomycin also induce apparent DNA double strand breaks, although the ratios of double to single strand breaks vary from those produced by X-ray. The introduction of double strand cuts by HpA I restriction endonuclease in DNA lysed on filters results in a rapid rate of elution under neutral conditions, implying that the method can detect double strand breaks if they exist in the DNA. The eluted DNA bands with a double stranded DNA marker in cesium chloride. This evidence suggests that the assay detects DNA double strand breaks. L1210 cells are shown to rejoin most of the DNA double strand breaks induced by 5-10 krad of X-ray with a half-time of about 40 minutes.     

Mechanisms of clastogen-induced chromosomal aberrations: A critical review and description of a model based on failures of tethering of DNA strand ends to strand-breaking enzymes

Most theories of the mechanisms of chromosomal aberrations involve the concepts of clastogens directly acting on DNA to produce strand breaks, and subsequently, the survival of these directly caused DNA strand breaks – or misrepairs of them – through to metaphase when they appear as chromosomal ‘breaks’ or translocations. Nevertheless, various observations are inconsistent with these theories such as the fact that many chemical clastogens (e.g. caffeine, acridines) do not covalently react with DNA, while almost all of the chemical clastogens (e.g. alkylating agents) which do react covalently with DNA, do not directly cause DNA strand breaks. This paper reviews the ‘direct-clastogen damage to DNA’ theories, and the phenomenology of chromosomal aberrations which are inconsistent with them. Then the theory is considered that the breaks in chromosomes seen at metaphase and anaphase are not the survivors of DNA breaks directly induced by clastogens, but rather derive from breaks created by the enzymes which repair damaged DNA. After that, newer knowledge is reviewed that (i) strand breaks are created during normal DNA unravelling (by topoisomerases), during DNA synthesis, and during DNA repairs, and these breaks can be single- or double-stranded, (ii) breaks variously associated with unravelling, synthesis and repair can occur ‘anywhere, anytime’ (pre-synthesis, synthesis or post-synthesis) in the cell cycle, and (iii) the enzyme assemblies for DNA unravelling, synthesis and repair which make and religate the breaks must be non-covalently tethered to the ends of the DNA strands while the breaks created by the enzymes are in existence. It is then suggested that all the morphological types and other phenomena of chromosomal aberrations can be explained by aspects, mechanisms and effects of failures of this tethering function. Circumstances involving the basic mechanism (failure of DNA-end-tethering function while enzyme-created breaks are in existence) are described which might result in ‘gaps’, translocations (‘exchanges’), complex lesions such as ‘triradials’, as well as in ‘minutes’, amplifications and inversions. Predictions are made concerning likely results in various suggested studies including those involving sensitive assays for DNA-end-to-enzyme tethering functions in vitro.     

Living with DNA Breaks is an Everyday Reality for Cells Adapted to High NaCl

A rapid, coordinated response to DNA breaks, including activation of cell cycle checkpoints and initiation of accurate DNA repair is believed to be necessary to maintain genomic integrity and prevent accumulation of mutations. That is why it was so unexpected to discover recently that in the mouse renal inner medulla the otherwise healthy cells contain numerous DNA breaks, yet they survive and function adequately. The DNA breaks in the renal inner medulla are caused by the high NaCl concentrations to which the cells are constantly exposed as a consequence of the urinary concentrating mechanism. Cells adapted to high NaCl in cell culture also contain many DNA breaks. The DNA breaks do not trigger cell cycle arrest or cause apoptosis, and the cells safely proliferate rapidly despite their presence. Further, high NaCl inhibits the activity of key components of the classical DNA damage response such as Mre11, chk1 and H2AX. In order to explain why the DNA breaks do not cause disabling mutations, oncogenic transformations and/or apoptosis we speculate that in the presence of high NaCl there might be alternative DNA damage response pathways or special ways of coping with DNA damage.     

The role of short-lived DNA lesions in the production of chromosome-exchange aberrations

Human lymphocytes were treated with combined UVC radiation and X-rays or they were X-irradiated and incubated for 60–90 min in the presence of DNA-repair inhibitor ara-C. The X-ray induced chromosome exchange aberration yield was enhanced both by UVC and ara-C by approximately a factor of two in the linear (low dose) portion of the dose-response curve. The enhancement was small in the dose squared (high dose) portion where previous dose-fractionation experiments have shown that X-ray-induced lesions leading to aberrations exist for several hours. The yield of aberrations in lymphocytes incubated after irradiation in the presence of ara-C reaches a saturation level almost immediately after irradiation (5–15 min). These cytogenetic observations together with a previous finding (Holmberg and Strausmanis, 1983) give direct and indirect evidence that the enhanced aberration yield is due to short-lived DNA breaks formed immediately after X-irradiation.

Measurements on the repair kinetics of the DNA breaks induced by X-irradiation show that ara-C strongly impairs the repair of short-lived X-ray-induced DNA breaks. It was also observed that the DNA breaks generated after UVC irradiation occur almost immediately after irradiation and the level of these transient DNA breaks reaches saturation even for short incubation times. Thus, the repair of these breaks can compete with the repair of short-lived X-ray-induced DNA-breaks in combined irradiation with UVC and X-rays.

The experimental results can be explained on the assumption that X-ray-induced aberrations originate from exchange complexes formed in interactions between both short-lived DNA breaks. The short-lived DNA breaks give rise to exchange complexes mainly within single ionization tracks where the DNA breaks are close together. The time between irradiation and exchange complex formation is of the order of 5–15 min within such a track, and short-lived breaks might be repaired before complexes have been formed. If the DNA repair of these breaks is delayed by UVC or ara-C treatment this results in a higher probability of exchange-complex formation. In contrast, interactions between breaks in different tracks originate from long-lived DNA breaks and the probability for complex formation from these breaks is not markedly affected by UVC or ara-C.     

Living with DNA breaks is an everyday reality for cells adapted to high NaCl

A rapid, coordinated response to DNA breaks, including activation of cell cycle checkpoints and initiation of accurate DNA repair is believed to be necessary to maintain genomic integrity and prevent accumulation of mutations. That is why it was so unexpected to discover recently that in the mouse renal inner medulla the otherwise healthy cells contain numerous DNA breaks, yet they survive and function adequately. The DNA breaks in the renal inner medulla are caused by the high NaCl concentrations to which the cells are constantly exposed as a consequence of the urinary concentrating mechanism. Cells adapted to high NaCl in cell culture also contain many DNA breaks. The DNA breaks do not trigger cell cycle arrest or cause apoptosis, and the cells safely proliferate rapidly despite their presence. Further, high NaCl inhibits the activity of key components of the classical DNA damage response such as Mre11, chk1 and H2AX. In order to explain why the DNA breaks do not cause disabling mutations, oncogenic transformations and/or apoptosis we speculate that in the presence of high NaCl there might be alternative DNA damage response pathways or special ways of coping with DNA damage.     

Characterization of DNA damage induced by 3,4-estrone-o-quinone in human cells.

The DNA damage induced in a human breast cancer cell line treated with 1,5 (10)-estradiene-3,4,17-trione (3,4-estrone-o-quinone; 3,4-EQ) has been measured qualitatively and quantitatively. Single-strand (ss) but not double-strand (ds) DNA breaks were formed in MCF-7 cells treated with 3,4-EQ. The ss DNA breaks formed in MCF-7 cells were partially repaired after incubation of cells in 3,4-EQ-free media for 2 and 4 h (i.e. 33 and 23% repair, respectively, as compared to the ss DNA breaks in cells after a 1-h exposure to 3,4-EQ without a recovery period). The formation of interstrand DNA cross-links was demonstrated in MCF-7 cells exposed to the bifunctional alkylating agent, mitomycin C, but not in those exposed to 3,4-EQ. Protein-linked DNA breaks were detected in MCF-7 cells after exposure to camptothecin and etoposide but not 3,4-EQ, suggesting that the ss DNA breaks induced by 3,4-EQ are unlikely to be mediated via topoisomerases. The induction of ss DNA breaks was detected in the estrogen receptor-negative cell line, BT-20, after exposure to 3,4-EQ. Furthermore, excess estradiol in culture media did not prevent 3,4-EQ-induced ss DNA breaks, suggesting that the DNA damage was not mediated via the estrogen receptor. Evaluation of the newly synthesized quinone analogue, 5,6,7,8-tetrahydro-1-2-naphthoquinone, in the ss DNA breakage assay revealed that the A and B ring moiety of 3,4-EQ is sufficient to produce ss DNA breaks in MCF-7 cells.     

Formation, resealing and persistence of DNA breaks produced by 4-demethoxydaunorubicin in P388 leukemia cells

The formation and disappearance of DNA single-strand breaks (SSB) produced by 4-demethoxydaunorubicin (4-dmDR) in P388 murine leukemia cells and in a resistant subline were examined by alkaline elution methods in relation to cellular pharmacokinetics. DNA strand breaks produced by this intercalating agent were essentially DNA lesions mediated by topoisomerase II, even at very high drug concentrations, since they were detected as protein-associated breaks by filter elution. Similarly, the appearance of delayed DNA breaks in cells exposed to high concentrations, following drug removal, showed predominance of protein-associated breaks, thus supporting a similar mechanism of breakage induction. This finding indirectly suggests that, in this experimental model, free radical production makes little (if any) contribution to DNA damage, and also that DNA effects are not the consequence of early cell death. In contrast to a rapid disappearance of protein-associated strand breaks produced by intercalating agents and topoisomerase II inhibitors of different classes, DNA breaks induced by low concentrations of the anthracycline derivative are only partially reversible following drug removal, but they persisted and even increased with high concentrations. Thus, not only the extent of DNA breaks but also their persistence may contribute to the cytotoxic potency of anthracyclines. The importance of DNA lesions to cytotoxic action of the anthracycline is also emphasized by drug effect on the resistant line. A negligible effect on DNA of resistant cells was detected at drug concentrations lethal to sensitive cells. However, exposure to equitoxic drug concentrations resulted in a comparable amount of DNA breaks in sensitive and resistant cells. Although faster DNA rejoining in resistant cells may be in part attributable to increased efflux of drug, no correlation exists between cell drug accumulation and extent of DNA lesions. With equitoxic drug concentrations cellular drug content was higher in resistant cells, suggesting an intrinsic insensitivity of this variant to DNA cleavage effects of the anthracycline.     

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.     

Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of cytotoxic action of 5-fluorodeoxyuridine (FdUrd) in mouse FM3A cells was investigated. We observed the FdUrd-induced imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools and subsequent double strand breaks in mature DNA, accompanied by cell death. The imbalance of dNTP pools was maximal at 8 h after 1 microM FdUrd treatment; a depletion of dTTP and dGTP pools and an increase in the dATP pool were observed. The addition of FdUrd in culture medium induced strand breaks in DNA, giving rise to a 90 S peak by alkaline sucrose gradient sedimentation. The loss of cell viability and colony-forming ability occurred at about 10 h. DNA double strand breaks as measured by the neutral elution method were also observed in FdUrd-treated cells about 10 h after the addition. These results lead us to propose that DNA double strand breaks play an important role in the mechanism of FdUrd-mediated cell death. A comparison of the ratio of single and double strand breaks induced by FdUrd to that observed following radiation suggested that FdUrd produced double strand breaks exclusively. Cycloheximide inhibited both the production of DNA double strand breaks and the FdUrd-induced cell death. An activity that can induce DNA double strand breaks was detected in the lysate of FdUrd-treated FM3A cells but not in the untreated cells. This suggests that FdUrd induces the cellular DNA double strand breaking activity. The FdUrd-induced DNA strand breaks and cell death appear to occur in the S phase. Our results indicate that imbalance of the dNTP pools is a trigger for double strand DNA break and cell death.     

A method for measuring the number of single- and double-strand breaks introduced into DNA molecules by the action of deoxyribonucleases.

A method is described for measuring the average number of nuclease-induced single- and double-strand breaks per DNA molecule. The procedure involves measuring the weight-average molecular weight of DNase I-digested DNA under neutral and alkaline conditions. A statistical equation is used to calculate the number of breaks per single- or double-stranded DNA molecule from the respective weight-average molecular weights. Enzymatic incorporation of³²P into the 5′-OH ends of DNase I-induced breaks gave an independent measurement of the number of breaks per DNA molecule. Results obtained by the two different methods were in good agreement. In agreement with earlier reports we find that magnesium-activated DNase catalyzes a high frequency of single-strand breaks in DNA. The frequency of double-strand breaks is low, but significantly higher than can be explained by random accumulation of single-strand breaks. Our data suggest that the frequency of double-strand scission is affected by DNase-metal ion interactions.     

Meiotic DNA breaks associated with recombination in S. pombe

In the fission yeast Schizosaccharomyces pombe, we have detected prominent DNA breaks that appeared shortly after premeiotic DNA replication. These breaks, like meiotic recombination, required the products of the six rec genes tested. Prominent breaks were detected at widely separated sites, about 100-300 kb apart, equivalent to about 50-150 sites per genome or approximately the number of meiotic recombination events. Certain features of these breaks are similar to those in the distantly related yeast Saccharomyces cerevisiae, the only other organism in which meiotic DNA breaks have been reported. Other features, however, appear to be different. These results suggest that, although DNA breaks may be a general feature of meiotic recombination, the breaks in S. pombe may play a role different from those in S. cerevisiae.     

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.