Qualitative and quantitative aspects of intercalator-induced DNA strand breaks.

The intercalating agents, adriamycin and ellipticine, were previously found to produce DNA strand breaks associated with DNA-protein crosslinks in mouse leukemia L1210 cells. The current work explores the nature of the agents that produce this effect and the quantitative relationship between the breaks and crosslinks. The protein-associated DNA breaks were produced by a wide variety of intercalators in addition to the above-mentioned compounds: actinomycin D, daunoycin, ethidium and lucanthone (miracil D). Treatment with several drugs that bind to DNA without intercalation, or that inhibit DNA synthesis without binding to DNA, did not cause DNA breaks. The strand break and crosslink frequencies were quantitated by means of alkaline elution methods. The strand break and crosslink frequencies were found to be within a factor of 2 of each other over a range of concentrations of adriamycin and ellipticine. It is proposed that intercalation-induced distortion of the DNA helix leads to strand scission by a nuclease which becomes bound to one terminus of the break so as to form a DNA-protein crosslink.     

Changes in deoxyribonucleic acid linking number due to treatment of mammalian cells with the intercalating agent 4'-(9-acridinylamino)methanesulfon-m-anisidide

Treatment of mammalian cells with DNA intercalating agents produces protein-associated DNA strand breaks. These breaks have been proposed to represent the action of a topoisomerase, which would alter the DNA linking number. Changes in DNA linking number in cells treated with the intercalating agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) were studied by ethidium titration of nucleoid sedimentation. m-AMSA treatment was found to produce an increase in DNA linking number. Previously, we had proposed that intercalator-induced protein-associated DNA breaks act to reduce DNA torsional strain that results from the intercalator-induced decrease in DNA twist. In such a model, linking number would be expected to decrease. The finding that the DNA linking number increased following m-AMSA treatment suggests that intercalators may block enzymes that normally decrease linking number. Such enzymes would have DNA gyrase like properties. Consistent with this possibility, a DNA gyrase inhibitor, novobiocin, inhibited the restoration of normal linking number and, to a lesser degree, the reversal of protein-associated strand breaks after removal of intercalator.     

Ellipticine-induced protein-associated DNA breaks in isolated L1210 nuclei

DNA intercalating agents have been found to produce protein-associated DNA strand breaks in mammalian cells. As a first step towards a subcellular system for the study of this reaction, we demonstrate that the reaction can take place in isolated cell nuclei. Ellipticine induces in these nuclei DNA strand breaks and stable DNA-protein complexes. Complexes and breaks are present in equivalent amounts. DNA breaks are revealed only if protein-mediated DNA adsorption to filters is abolished. These findings make it unlikely that similar effects observed in cells in culture after treatment with intercalating agents are caused by metabolically activated drugs.     

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.     

Effects of the DNA intercalators 4'-(9-acridinylamino)methanesulfon-m-anisidide and 2-methyl-9-hydroxyellipticinium on topoisomerase II mediated DNA strand cleavage and strand passage

DNA topoisomerase II is believed to be the enzyme that produces the protein-associated DNA strand breaks observed in mammalian cell nuclei treated with various intercalating agents. Two intercalators--4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA, amsacrine) and 2-methyl-9-hydroxyellipticinium (2-Me-9-OH-E+)--differ in their effects on protein-associated double-strand breaks in isolated nuclei. m-AMSA stimulates their production at all concentrations, whereas 2-Me-9-OH-E+ stimulates at low concentrations and inhibits at high concentrations. We have reproduced these differential effects in experiments carried out in vitro with purified L1210 DNA topoisomerase II, and we have found that concentrations of 2-Me-9-OH-E+ above 5 microM prevent the trapping of DNA-topoisomerase II cleavable complexes irrespective of the presence of m-AMSA. It also stimulated topoisomerase II mediated DNA strand passage, again with or without inhibitory amounts of m-AMSA (this result suggests that extensive intercalation by 2-Me-9-OH-E+ destabilized the cleavable complexes). From these data, it is concluded that intercalator-induced protein-associated DNA strand breaks observed in intact eukaryotic cells and isolated nuclei are generated by DNA topoisomerase II and that intercalators can affect mammalian DNA topoisomerase II in more than one way. They can trap cleavable complexes and inhibit DNA topoisomerase II mediated DNA relaxation (m-AMSA and low concentrations of 2-Me-9-OH-E+) or destabilize cleavable complexes and stimulate DNA relaxation (high concentrations of 2-Me-9-OH-E+).     

DNA damage and cytotoxicity of nitracrine in cultured HeLa cells

The effects of nitracrine (1-nitro-9-(3,3-N,N-dimethylaminopropylamino)acridine on DNA of cultured HeLa cells were studied. DNA strand breakage and interstrand cross-linking as well as DNA-protein cross-linking were measured by means of an alkaline elution technique and were compared with the cytotoxic effect of the drug. Interstrand cross-links were not detectable in the concentration range that inhibited cell growth up to 99%. DNA single-strand breaks were found when cells were treated with highly cytotoxic doses of the drug. DNA breakage was not reparable and exhibited a tendency to increase during incubation after drug removal. The only chromatin lesion induced by sublethal doses of nitracrine were DNA-protein cross-links which persisted for 24 h after drug treatment. It is concluded that DNA breaks represent degraded DNA from dying cells, whereas DNA-protein cross-links are specific cellular lesions, which may be responsible for the cell-killing effect of nitracrine.     

DNA topoisomerase I as a site of action for 10-hydroxycamptothecin in human promyelocytic leukemia cells

We investigated the antiproliferative effect of 10-hydroxycamptothecin (HCPT), an alkaloid isolated from Camptotheca acuminata, on the human promyelocytic leukemia cell line, HL-60, and a 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)-resistant mutant, HL-60/m-AMSA. Using trypan blue dye exclusion and colony formation, doses of HCPT ranging from 0.01 to 1 microM progressively inhibited growth in both cell lines in a concentration-dependent manner. A minimal cross-resistance, approximately five-fold, between the wild-type and resistant cells was observed. Using the technique of alkaline elution, HCPT produced DNA single-strand breaks and protein-associated DNA strand cleavage in HL-60 and HL-60/m-AMSA cells. Quantitative analysis of drug-induced protein-DNA complexes was performed using sodium dodecyl sulfate-potassium chloride precipitation. In both cell lines, a good correlation with HCPT-induced cytotoxicity was observed. Similar results were achieved in wild-type cells treated with m-AMSA. Enzyme activity was measured in nuclei isolated from HL-60 and HL-60/m-AMSA cells, and in each case HCPT inhibited topoisomerase I activity to the same extent. The data suggest that the principle mechanisms for HCPT-induced cytotoxicity in HL-60 and HL-60/m-AMSA cells are inhibition of DNA topoisomerase I and production of protein-associated DNA strand breaks.     

Characterization of DNA lesions produced by HgCl2 in cell culture systems

HgCl2 is extremely cytotoxic to Chinese hamster ovary (CHO) cells in culture since a 1-h exposure to a 75- microM concentration of this compound reduced cell plating efficiency to 0 and cell growth was completely inhibited at 7.5 microM . The level of HgCl2 toxicity depended upon the culture incubation medium and has previously been shown to be inversely proportional to the extracellular concentration of metal chelating amino acids such as cysteine. Thus, HgCl2 toxicity in a minimal salts/glucose maintenance medium was about 10-fold greater than the toxicity in McCoy's culture medium. The HgCl2 toxicity in the latter medium was 3-fold greater than that in alpha-MEM which contains more of the metal chelating amino acids. When cells were exposed to HgCl2 there was a rapid and pronounced induction of single strand breaks in the DNA at time intervals and concentrations that paralleled the cellular toxicity. The DNA damage was shown to be true single strand breaks and not alkaline sensitive sites or double strand breaks by a variety of techniques. Consistent with the toxicity of HgCl2, the DNA damage under an equivalent exposure situation was more pronounced in the salts/glucose than in the McCoy's medium and more striking in the latter medium than in alpha-MEM. Most of the single strand breaks occurred within 1 h of exposure to the metal. We believe that the DNA damage caused by HgCl2 leads to cell death because the DNA single strand breaks are not readily repaired. DNA repair activity measured by CsCl density gradient techniques was elevated above the untreated levels at HgCl2 concentrations that produced little measurable binding of the metal to DNA or few single strand breaks assessed by the alkaline elution procedure. DNA repair activity decreased at HgCl2 concentrations that produced measurable DNA binding and single strand breaks. These irreversible interactions of HgCl2 with DNA may be responsible for its cytotoxic action in cells.     

Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.     

Formation and rejoining of deoxyribonucleic acid double-strand breaks induced in isolated cell nuclei by antineoplastic intercalating agents

The biochemical characteristics of the formation and disappearance of intercalator-induced DNA double-strand breaks (DSB) were studied in nuclei from mouse leukemia L1210 cells by using filter elution methodology [Bradley, M. O., & Kohn, K.W. (1979) Nucleic Acids Res. 7, 793-804]. The three intercalators used were 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), 5-iminodaunorubicin (5-ID), and ellipticine. These compounds differ in that they produced predominantly DNA single-strand breaks (SSB) (m-AMSA) or predominantly DNA double-strand breaks (ellipticine) or a mixture of both SSB and DSB (5-ID) in whole cells. In isolated nuclei, each intercalator produced DSB at a frequency comparable to that which is produced in whole cells. Moreover, these DNA breaks reversed within 30 min after drug removal. It thus appeared that neither ATP nor other nucleotides were necessary for intercalator-dependent DNA nicking-closing reactions. The formation of the intercalator-induced DSB was reduced at ice temperature. Break formation was also reduced in the absence of magnesium, at a pH above 6.4 and at NaCl concentrations above 200 mM. In the presence of ATP and ATP analogues, the intercalator-induced cleavage was enhanced. These results suggest that the intercalator-induced DSB are enzymatically mediated and that the enzymes involved in these reactions can catalyze DNA double-strand cleavage and rejoining in the absence of ATP, although the occupancy of an ATP binding site might convert the enzyme to a form more reactive to intercalators. Three inhibitors of DNA topoisomerase II--novobiocin, nalidixic acid, and norfloxacin--reduced the formation of DNA strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)     

The contribution of alkylation to the activity of quinone antitumor agents

Studies have shown that the quinone group can produce tumor cell kill by a mechanism involving active oxygen species. This cytotoxic activity can be correlated with the induction of DNA double strand breaks and is enhanced by the ability of the quinone compound to bind to DNA by alkylation. The cytotoxic activity and the production of DNA damage by model quinone antitumor agents were compared in L5178Y cells, sensitive and resistant to alkylating agents, to assess the contribution of alkylation to the activity of these agents. The resistant L5178Y/HN2 cells were found to be two fold and six fold more resistant to the alkylating quinones, benzoquinone mustard and benzoquinone dimustard, respectively, than parent L5178Y cells. In contrast, the L5178Y/HN2 cells showed no resistance to the nonalkylating quinones, hydrolyzed benzoquinone mustard and bis(dimethylamino)benzoquinone. The alkylating quinones produced approximately two fold less cross-linking in L5178Y/HN2 cells compared with L5178Y sensitive cells. DNA double strand break formation by hydrolyzed benzoquinone mustard and bis(dimethylamino)benzoquinone was not significantly different in sensitive and resistant cells. However, the induction of double strand breaks by the alkylating quinones benzoquinone mustard and benzoquinone dimustard was reduced by 5-fold and 15-fold, respectively, in L5178Y/HN2 cells. These results show that the alkylating activity of the alkylating quinones cannot directly explain all of the enhanced cytotoxic activity of these agents. Furthermore, they provide strong evidence that the enhanced formation of DNA double strand breaks by alkylating quinone agents is directly related to the ability of these agents to bind to DNA. This increased formation of strand breaks may account for the enhanced cytotoxic activity of the alkylating quinones.     

DNA damage induced by carcinogenic lead chromate particles in cultured mammalian cells.

Particulate lead chromate is a highly water-insoluble cytotoxic and carcinogenic agent, but its mechanism of action remains obscure. We investigated its effects on DNA damage in CHO cells after a 24-h exposure using alkaline or neutral filter elution and cytogenetic studies. Concentrations (0.08, 0.4 and 0.8 micrograms/cm2), which reduced the colony-forming efficiency of CHO cells to 94, 50 and 10%, respectively, produced dose-dependent DNA single-strand breaks and DNA-protein crosslinks, but no DNA double-strand breaks or DNA-DNA crosslinks were observed. The single-strand breaks were absent from cells given a 24-h recovery period after removal of the treatment medium, even though most of the particles remained adhered to cells and to the culture dish. In contrast, both the DNA-protein crosslinks and chromosomal aberrations persisted even after the 24-h recovery period. These results suggest that the mechanism of the particle-induced early DNA single-strand breaks may be different from DNA-protein crosslinks and the lesions leading to chromosomal aberrations, or alternatively, that the repair of single-strand breaks is more efficient than the repair of DNA-protein crosslinks in the unavoidable continuing presence of carcinogen. These results also suggest that the chromosome damage may be related to the persistent DNA-protein crosslinks, and further confirm the genotoxic activity of carcinogenic lead chromate particles.     

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.     

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.     

125I-induced DNA double strand breaks: use in calibration of the neutral filter elution technique and comparison with X-ray induced breaks

The neutral filter elution assay, for measurement of DNA double strand breakage, has been calibrated using mouse L cells and Chinese hamster V79 cells labelled with [125I]dUrd and then held at liquid nitrogen temperature to accumulate decays. The basis of the calibration is the observation that each 125I decay, occurring in DNA, produces a DNA double strand break. Linear relationships between 125I decays per cell and lethal lesions per cell (minus natural logarithm survival) and the level of elution, were found. Using the calibration data, it was calculated that the yield of DNA double strand breaks after X-irradiation of both cell types was from 6 to 9 X 10(-12) DNA double strand breaks per Gy per dalton of DNA, for doses greater than 6 Gy. Neutral filter elution and survival data for X-irradiated and 125I-labelled cells suggested that the relationships between lethal lesions and DNA double strand breakage were significantly different for both cell types. An attempt was made to study the repair kinetics for 125I-induced DNA double strand breaks, but was frustrated by the rapid DNA degradation which occurs in cells that have been killed by the freezing-thawing process.     

Nuclear topoisomerase II levels correlate with the sensitivity of mammalian cells to intercalating agents and epipodophyllotoxins

We have investigated the biochemical basis for the hypersensitivity to intercalating agents and epipodophyllotoxins of a Chinese hamster cell mutant, ADR-1. More topoisomerase II-induced DNA strand breaks are accumulated by ADR-1 than by parental CHO-K1 cells following exposure to the intercalating agent amsacrine. Levels of induced DNA strand breaks correlate with cell killing. Topoisomerase II activity is elevated in ADR-1 cells as a consequence of an increased cellular level of topoisomerase II protein. We have studied the phenotype of cell hybrids generated by fusing parental and mutant cells. The hybrid ADR-1/CHO-K1 exhibits normal levels of resistance to amsacrine and expresses the lower, parental level of topoisomerase II. These results provide additional evidence that topoisomerase II mediates the cytotoxic action of intercalating agents and epipodophyllotoxins and suggest that the intracellular level of topoisomerase II is an important determinant of cellular sensitivity to these drugs. This has implications for antitumor therapy. ADR-1 cells provide a model system for studying the effects of topoisomerase II overproduction on cell proliferation and chromosome organization.     

DNA-protein cross-links induced by nickel compounds in intact cultured mammalian cells

The carcinogenic activity of crystalline NiS has been attributed to phagocytosis and intracellular dissolution of the particles to yield Ni2+ which is thought to enter the nucleus and damage DNA. In this study the extent and type of DNA damage in Chinese hamster ovary CHO cells treated with various nickel compounds was assessed by alkaline elution. Both insoluble (crystalline NiS) and soluble (NiCl2) nickel compounds induced single strand breaks and DNA protein cross-links. The single strand breaks were repaired relatively quickly but the DNA-protein cross-links were present and still accumulating 24 h after exposure to nickel. Single strand breakage occurred at both non-cytotoxic and cytotoxic concentrations of nickel, however, DNA-protein cross-linking was absent when cells were exposed to toxic nickel levels. The concentration of nickel that induced DNA-protein cross-linking correlated with those metal concentrations that reversibly inhibited cellular replication.     

Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O6-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase beta were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.     

Reconstitution of intercalator-induced DNA scission by an active component from nuclear extracts

Treatment with intercalating agents causes formation of protein-associated DNA breaks in mammalian cells in culture and in the nuclei isolated from these cells. We found that this effect, when induced by the intercalator m-AMSA, required a component which could be dissociated from nuclei by 0.3 M NaCl. The effect was restored by combining the extracted nuclei with the nuclear extract. The active component of the extract eluted in gel filtration at a point corresponding to a molecular weight of 800 000. During its reaction with DNA, DNA-protein links and DNA breaks appeared in approximately equal frequencies. In this respect the reaction stimulated by m-AMSA resembled the reaction of a topoisomerase with DNA. However, intercalator-stimulated formation of protein-associated DNA breaks differed from the activity of the nuclear topoisomerase I in that there was a different optimum salt concentration and a different apparent molecular weight.     

Apurinic site induction in the DNA of cells heated at hyperthermic temperatures

The induction of DNA damage in cells heated at hyperthermic (43-48 degrees C) temperatures was determined by alkaline filter elution and alkaline sucrose gradient-sedimentation analysis of cell DNA denatured at pH 13.0. A class of DNA lesion which converted to strand breaks during denaturation of DNA at pH 13.0 was produced randomly throughout the cell DNA at temperatures as low as 43 degrees C. Induction of this lesion occurred with a T0 of 90 and 10 min at 45 and 48 degrees C, respectively. We estimate that these pH 13.0-detectable DNA lesions are produced in the cell DNA with a frequency of approximately 75 and 660 per min of heating at 45 and 48 degrees C, respectively. Since the lesions were quantitatively converted to DNA strand breaks at pH 13.0 with a half-time of 30 min, or less, we suggest that these pH 13.0-detectable DNA lesions are heat-induced, abasic DNA sites. The induction of these lesions does not appear to be directly involved in the initial heat-induced inhibition of DNA synthesis. The presence of these lesions cannot be excluded as an explanation for the long-term inhibition of replicon initiated in heated cells.