Alkaline Sucrose Gradient Centrifugation Research Articles

Ionizing irradiation effects on S-phase in checkpoint mutants of the yeast Saccharomyces cerevisiae.

In mammalian cells, gamma-irradiation activates checkpoint controls to delay entry into, or passage through S-phase, while chronic exposure to methyl methanesulfonate or hydroxyurea causes a similar delay in yeast. In yeast, at least five genes are involved: RAD9, RAD17, RAD24, RAD53 and MEC1, a homologue of ATM. Here, using flow cytometry analysis and alkaline sucrose gradient centrifugation of labeled, newly made DNA, we demonstrate, in synchronized RAD wild-type Saccharomyces cerevisiae cells, that: (1) gamma-irradiation at START delays entry into S-phase, (2) irradiation shortly before or during early S-phase delays completion of S-phase and (3) the latter response is largely a consequence of replicon initiation inhibition. The delay produced by irradiation during early S-phase depends on the function of the checkpoint genes RAD9, RAD17, RAD24, RAD53, MEC1 and MEC3. However, at least four, RAD17, RAD53, MEC1, MEC3, are not needed to delay S-phase progression when cells are irradiated shortly before S-phase begins.

Ultrasensitive detection of DNA damage by the combination of the comet and TUNEL assays.

The assessment of cellular DNA damage is crucial in many areas of biology including immunology, developmental biology, aging, cancer, and environmental science. A variety of experimental techniques including alkaline sucrose gradient centrifugation, alkaline elution, nucleoid sedimentation, viscoelastic measurements of DNA, alkaline unwinding, and gel electrophoresis (, , , ) have been developed to detect DNA damage in large cell populations. However, these methods can only detect the average amount of DNA damage over millions of cells. These approaches do not reveal the heterogeneity of DNA damage within a sample; some cells may experience extensive damage whereas others may display no damage at all. Moreover, one might want to analyze the amount of DNA damage in variety of cell subpopulations within a sample. In more specialized applications (e.g., a specific target cell undergoing immunologic recognition and destruction) the extent of DNA damage in a particular cell may be required. Furthermore, there are some clinical experiments where the amount of sample is severely limited (e.g., biopsy material) and therefore large-scale assays are impossible. Thus, there are many biological circumstances that require the use of small cell samples. To extract DNA damage information from these samples, techniques that rely upon the evaluation of DNA damage at the level of single cells is required. The single cell gel electrophoresis (SCGE) or “comet” assay is the most widely applied method for the detection of DNA damage in single cells. In this approach damaged DNA in individual cells is electrophoresed away from a nucleus into an agarose gel followed by staining.

The catalytic subunit DNA-dependent protein kinase (DNA-PKcs) facilitates recovery from radiation-induced inhibition of DNA replication.

Exposure of cells to ionizing radiation inhibits DNA replication in a dose-dependent manner. The dose response is biphasic and the initial steep component reflects inhibition of replicon initiation thought to be mediated by activation of the S-phase checkpoint. In mammalian cells, inhibition of replicon initiation requires the ataxia telagiectasia mutated ( ATM ) gene, a member of the phosphatidyl inositol kinase-like (PIKL) family of protein kinases. We studied the effect on replicon initiation of another member of the PI-3 family of protein kinases, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) by measuring either total DNA synthesis, or size distribution of nascent DNA using alkaline sucrose gradient centrifugation. Exposure of human cells proficient in DNA-PKcs (HeLa or M059-K) to 10 Gy inhibited replicon initiation in a time-dependent manner. Inhibition was at a maximum 1 h after irradiation and recovered at later times. Similar treatment of human cells deficient in DNA-PKcs (M059-J) inhibited replicon initiation to a similar level and with similar kinetics; however, no evidence for recovery, or only limited recovery, was observed for up to 8 h after irradiation. In addition a defect was observed in the maturation of nascent DNA. Similarly, a Chinese hamster cell line deficient in DNA-PKcs (irs-20) showed little evidence for recovery of DNA replication inhibition up to 6 h after irradiation, whereas the parental CHO cells showed significant recovery and an irs-20 derivative expressing the human DNA-PKcs complete recovery within 4 h. Normal kinetics of recovery were observed in xrs-5 cells, deficient in Ku80; in 180BR cells, deficient in DNA ligase IV; as well as XR-1 cells, deficient in XRCC4, an accessory factor of DNA ligase IV. Since all these cell lines share the DNA double strand break rejoining defect of M059-J and irs20 cells, the lack of recovery of DNA replication in the latter cells may not be attributed entirely to the prolonged presence of unrepaired DNA dsb. We propose that DNA-PKcs, in addition to its functions in the rejoining of DNA dsb and in DNA replication, also operates in a pathway that in normal cells facilitates recovery of DNA replication after irradiation.

DNA damage and cell killing by camptothecin and its derivative in human leukemia HL-60 cells.

Camptothecin (CPT) has heen recognized as a topoisomerase I (Topo I) inhibitor. However, the mechanism of cytotoxicity of this agent remains unknown. In the present study, we analyzed the kinetics of Topo I‐mediated DNA single‐strand breaks and internucleosomal DNA cleavage produced by CPT and its derivative, 7‐ethyl‐10‐hydroxycamptothecin (SN‐38), in HL‐60 cells. DNA single‐ strand breaks were detected using alkaline sucrose gradient centrifugation when HL‐60 cells were incubated with 10 μM CPT or 10 μM SN‐38 for 30 min. These DNA single‐strand breaks were rapidly repaired after drug removal, while the cytotoxic action of these drugs was sustained. Treatment of HL‐60 cells with CPT or SN‐38 for 3 h produced extensive degradation of DNA. Agarose gel electrophoresis showed a ladder of DNA fragments consisted of multimers of approximately 200 base pairs, characteristic of apoptosis. Interestingly, this type of DNA fragmentation was also induced within 4 h after repair of DNA single‐strand breaks, and subsequently loss of cell viability was observed. When zinc ion, a potent inhibitor of endonuclease, was added to drug‐free medium after treatment with CPT or SN‐38, internucleosomal DNA cleavage was abolished. Furthermore, addition of zinc ion reduced the loss of cell viability. These data suggest that Topo I‐mediated DNA single‐strand breaks may be necessary but are not sufficient for cell death, and the endonuclease involved in induction of internucleosomal DNA cleavage may play an important role in HL‐60 cell death induced by Topo I inhibitor.

Cycloheximide inhibits cellular, but not SV40, DNA replication

We have prepared extracts from cycloheximide-treated cells for the study of simian-virus-40 (SV40)-DNA replication in vitro. When supplemented with the viral initiator protein (large T antigen), these extracts fully supported SV40-DNA replication. We also determined that SV40-DNA replication in vivo is much more resistant to cycloheximide than cellular DNA replication. SV40 encodes its own initiator protein, T antigen, which also functions as a DNA helicase, but depends on cellular functions for all additional replication reactions. Therefore, it appears to be quite likely that cycloheximide affects cellular DNA replication by blocking the synthesis of (a) cellular function(s) that is(are) performed by T antigen in SV40-DNA replication. Indeed, DNA fiber autoradiography and alkaline sucrose gradient centrifugation of pulse-labeled cellular DNA showed that cycloheximide treatment almost completely suppressed replicon initiation and reduced the rate of replication fork movement to about one third of the control.

Mapping Eukaryotic Replication OriginsIn Vivoby Size Analysis of Purified Nascent DNA Strands

A simple and efficient method for the mapping of eukaryotic replication origins was tested. The method is based on differential labeling of newly synthesized DNA with BrdUrd and subsequent separation of heavy nascent strands from parental DNA by conventional alkaline sucrose and neutral CsCl isopycnic gradient centrifugation. Purified nascent DNA is then size-fractionated on alkaline agarose gels and analyzed by sequential hybridization to specific probes of known location on the DNA segment of interest. Evaluation of the hybridization results allows: (i) determination of the direction of replication fork movement and (ii) location of the initiation site of DNA synthesis. Taking SV40 and polyoma virus as model systems, we demonstrate the feasibility of this procedure. It applicability to the location of chromosomal replication origins is discussed.

Repair of DNA Single-Strand Breaks in Lymphocytes from Alzheimer’s Disease Patients

We have studied DNA single-strand breaks (SSB) and their repair, after gamma-irradiation, in lymphocytes from patients with Alzheimer's disease (AD) and from normal, age-matched individuals, using alkaline sucrose gradient centrifugation. We have found that values for AD patients and normals are similar in each of the following: level of breaks in unirradiated cells, level in cells irradiated with 150 Gy and rate of repair. Also, preliminary results for young and old normal individuals showed no significant difference between their SSB levels in unirradiated cells, nor between levels in irradiated cells nor in repair rate. In all cases, post-irradiation repair appeared to be almost complete after 50 min incubation. We conclude that gamma-irradiation produces no gross difference in the initial number of SSB between lymphocytes from AD patients, old normals and young normals, and that their repair is equally proficient.

Arabinosylcytosine‐induced accumulation of DNA nicks in myotube nuclei detected by in situ nick translation

This laboratory has recently reported the occurrence of DNA nicking at the onset of terminal skeletal myogenesis by using the technique of in situ nick translation (Dawson and Lough: Dev. Biol., 127:362-367, 1988). Because 1-beta-D-arabinofuranosylcytosine (araC), a cytocidal agent that is routinely used to removed dividing fibroblasts from myogenic cultures, inhibits DNA repair, it was of interest to determine whether araC treatment resulted in an accumulation of the endogenously created nicks. Thus, we have assessed the accumulation of DNA nicks in myotube cells during a 20 hour araC treatment period at the onset of terminal myogenesis (44-64 hours in vitro) by using three techniques: alkaline sucrose gradient density centrifugation, kinetic in situ nick translation, and cellular in situ nick translation. Although alkaline sucrose gradient centrifugation revealed no detectable nicking after 20 hours, kinetic in situ nick translation analysis revealed subtle but significant increases in DNA nicks caused by araC within 7 hours of drug application, and a 1.5-fold increase in DNA repair sites after 20 hours of drug treatment. That these observations reflected nicking specifically in myotube nuclei was determined by immunocytochemical localization of nicked sites after repair with a biotinylated nucleotide analog (biotin-11-dUTP). The effects of araC were only incompletely reversible, whether or not the drug was removed from the cultures, within 2 days of the treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein-primed replication of bacteriophage PRD1 genome in vitro

A cell-free system has been developed from cells of an Escherichia coli strain, carrying cloned genes 1 and 8 of bacteriophage PRD1, that catalyzes protein-primed DNA synthesis. DNA synthesis in vitro is entirely dependent upon the addition of PRD1 DNA-protein complex as template, Mg 2+, and four deoxyribonucleoside triphosphates. No in vitro DNA synthesis was observed when deproteinized PRD1 DNA was used as template. The origin and direction of PRD1 DNA replication in vitro was determined by restriction enzyme analysis of 32P-labeled PRD1 DNA synthesized in this system. Replication starts at both ends of the linear PRD1 DNA template. Alkaline sucrose gradient centrifugation and agarose gel electrophoresis showed that full-length PRD1 DNA is synthesized in vitro. DNA synthesis in this system is inhibited by the drug aphidicolin. We also observed that dimethyl sulfoxide (DMSO) stimulates in vitro DNA synthesis, although it inhibits bacterial DNA polymerase.

Effects of the ortho-quinone and catechol of the antitumor drug VP-16-213 on the biological activity of single-stranded and double-stranded ΦX174 DNA

We have studied the effects of the recently reported two new metabolites of the antitumor agent VP-16-213, the ortho-dihydroxy derivative or catechol and the ortho-quinone, on the biological activity of single-stranded and double-stranded ΦX174 DNA, the binding of the metabolites to calf thymus DNA and the conversion of the catechol into the ortho-quinone. Evidence was obtained for the oxidation of the catechol into the ortho-quinone and for the fact that the ortho-quinone is the metabolite of VP-16-213 responsible for its binding to rat liver microsomal proteins. The catechol and ortho-quinone of VP-16-213 were found to bind 7–9 times more strongly to calf thymus DNA than VP-16-213 itself. In contrast to the parent compound VP-16-213, the catechol as well as the ortho-quinone inactivated both single-stranded (ss) and double-stranded (RF) biologically active ΦX174 DNA. The mean T 37-values for inactivation of ss and RF ΦX174 DNA by 2.2 × 10 −4M catechol at 37° and pH 7.4 were 96 and 640 min, respectively. Reduction of the ortho-quinone by NADPH cytochrome P-450 reductase resulted in formation of the catechol. The system ortho-quinone/NADPH cytochrome P-450 reductase inactivated ss ΦX174 DNA with a mean T 37-value of 454 min, and this inactivation was inhibited by DMSO. The mean T 37-value for inactivation of ss ΦX174 DNA by 1.8 × 10 −4 M ortho-quinone at 37° and pH 4.0 was 24 min. The chemical stability of the ortho-quinone and the extent of inactivation of ss ΦX174 DNA by the ortho-quinone were both pH-dependent: at higher pH the ortho-quinone was less stable and gave less inactivation of DNA. The aqueous decomposition product(s) of the ortho-quinone formed at pH 7.4 inactivated ss ΦX174 DNA with a mean T 37-value of 175 min. The rate of inactivation of RF ΦX174 DNA by the ortho-quinone at pH 4.0 was twice as low as the rate of inactivation of ss ΦX174 DNA: T 37 = 49 min. When using excision repair deficient E.coli mutants (uvrA − or uvrC −), a higher inactivation of RF ΦX174 DNA was found: T 37 = 29 min for uvrA − E. coli, indicating that a part of the DNA damage introduced by the incubation with ortho-quinone is removed by excision repair. Neutral and alkaline sucrose gradient centrifugation of ss ΦX174 DNA incubated with the ortho-quinone at pH 4.0 revealed the absence of DNA-breakage, suggesting that introduction of strand breaks and alkali-labile sites does not play a role in the inactivation of DNA by the ortho-quinone. Adduct-formation probably plays a role in the inactivation of DNA by this metabolite.

Effects of excess thymidylate on thymidylate low-requiring strains of Saccharomyces cerevisiae: high mutagenicity and absence of DNA strand breaks

dTMP exposure concentrations of 0.1 mM or higher are genotoxic in exponentially growing cells of thymidylate low-requiring mutants of Saccharomyces cerevisiae. Mutagenicity of excess dTMP is highest in an exposure concentration 10-fold of that needed for external supplementation of endogenously blocked thymidylate synthesis. Still higher dTMP concentrations are primarily cytotoxic. The canavanine forward-mutation system shows excess dTMP to be as potent a mutagen as irradiation by ultraviolet light. Mutagenicity of excess dTMP, however, differs from that of direct DNA-attacking mutagens in that it is highest in the absence of significant toxicity. Alkaline sucrose gradient centrifugation shows that excess dTMP does not induce significant numbers of DNA single- or double-strand breaks, while conditions of thymidylate deprivation lead to DNA-strand breaks and thymineless death.

Oxidative damage to DNA and replicative lifespan in cultured adrenocortical cells

Oxidative damage to DNA in cultured bovine adrenocortical cells was investigated by exposing cells to a sublethal concentration (10 μM) of cumene hydroperoxide under conditions previously shown to be deficient in the biological antioxidants selenium and α-tocopherol (vitamin E). DNA prepared from cells incubated for 4 h with 10 μM cumene hydroperoxide had a greater fraction showing resistance to S1 nuclease after denaturation and reassociation to a log C 0 t of −3. Cross-linking by cumene hydroperoxide was abolished in cells that had been grown in the presence of 20 nM selenite or 1 μM α-tocopherol for 96 h prior to peroxide addition, whereas such cells remained susceptible to crosslinking by nitrogen mustard. Extensive strand breaks in DNA from peroxide-treated cells as assessed by alkaline sucrose gradient centrifugation were greatly reduced in cells grown in selenite or α-tocopherol. Despite the evidence of damage to DNA, cumene hydroperoxide was not detectably mutagenic, in contrast to 5 μM methylnitronitrosoguanidine (MNNG), when assessed as the incidence of resistance to 25 μM ouabain. We confirmed that cumene hydroperoxide at >10 μM lowers cloning efficiency and that this is largely prevented by selenite or α-tocopherol. Additionally, selenite or α-tocopherol produced increased clonogenicity in cells not incubated with peroxide. To examine effects of the biological antioxidants on replicative lifespan, cells were grown continuously in fetal bovine serum (FBS), fibroblast growth factor (FGF), and selenite or α-tocopherol. Selenium increased replicative lifespan by 10–20% and α-tocopherol by 22–30%. Levels of DNA cross-links and strand breaks did not differ under any circumstances between early (second) passage and late (30th) passage cells. The experiments on replicative potential were all performed in the presence of FGF. When FGF was omitted from the culture medium, replicative lifespan was reduced by 85%. We conclude that types of damage to DNA resulting from peroxide exposure are not present in cells under standard culture conditions at early or late stages of the lifespan. Other work has noted a relationship between clonogenicity and replicative lifespan; thus, the increase in cloning efficiency seen with selenium and α-tocopherol may cause the observed slight increase in replicative lifespan. Oxidative damage does not appear to be a major determinant of cellular senescence in adrenocortical cells.

The in vivo effect of gossypol on macromolecular synthesis in rat testis

An in vitro study showed that in rat testis gossypol acts as a nonspecific inhibitor of macromolecular synthesis. In this report we describe the in vivo effect of gossypol on DNA, RNA, and protein synthesis in rat testis. Gossypol treatment of rats (20 mg/kg) for 6, 10 and 16 weeks resulted in partial inhibition of macromolecular synthesis. When status of infertility is achieved after 6 weeks, macromolecular synthesis of the spermatogenetic epithelium is not essentially affected by gossypol. In contrast to the previously described DNA strand breaking induction of gossypol in human fibroblasts in vitro, no DNA strand breaking in testes of gossypol-treated rats was found as established by alkaline sucrose gradient centrifugation.

Replication of phage phi 29 DNA with purified terminal protein and DNA polymerase: synthesis of full-length phi 29 DNA.

A system that replicates bacteriophage phi 29 DNA with protein p3 covalently attached to the two 5' ends, using as the only proteins the phi 29 DNA polymerase and the terminal protein, is described. Restriction analysis of the 32P-labeled DNA synthesized in vitro showed that all phi 29 DNA fragments were labeled. Analysis by alkaline sucrose gradient centrifugation of the DNA labeled during a 10-min pulse showed that, after a 20-min chase, about half of the DNA molecules had reached apparently full-length phi 29 DNA (approximately equal to 18,000 nucleotides). Ammonium ions strongly stimulated phi 29 DNA-protein p3 replication, the effect being due to stimulation of the initiation reaction. ATP was not required for phi 29 DNA-protein p3 replication, either in the initiation or elongation steps. The results show that the phi 29 DNA polymerase functions, not only in the formation of the p3-dAMP covalent initiation complex but also in the elongation of the latter, as the only DNA polymerase to produce full-length phi 29 DNA.

The giant (gt) mutants of Drosophila melanogaster alter DNA metabolism.

Abnormalities in DNA metabolism have been found in third-instar females of Drosophila melanogaster that are heteroallelic or homoallelic for X-chromosomal giant (gt) mutations. Analysis of DNA metabolism in larval brain ganglia was carried out using alkaline sucrose gradient centrifugation, incorporation assays and a neutral filter elution assay. These analyses show that gt stocks synthesize DNA of a reduced molecular weight, have an unusually high frequency of spontaneous single and double-strand breaks, and exhibit a reduction in the normal inhibition of DNA synthesis following treatment with UV and the carcinogen AAAF. These phenomena are not associated with a defect in the repair of X-ray induced DNA breaks nor are they accompanied by any alterations in chromosome stability. Analysis of homozygous 1(2)gl larvae also reveal that these phenomena are specific to the gt locus and are thus not attributable solely to an extended developmental program. These findings strengthen the suggestion that the genetic instability associated with gt is related to perturbations in chromosome metabolism (Green 1982).

Exposure of cdc8 homozygous diploids of Saccharomyces cerevisiae to nonpermissive temperature leads to an increase in mitotic conversion frequencies

In a diploid strain homozygous for the cdc8-1 mutation, a block in DNA synthesis caused by restrictive temperature resulted in a significant increase in the frequency of intragenic recombination at the HOM2 locus. Under restrictive conditions, incorporation of radioactivity into DNA was reduced to 2% of the control and alkaline sucrose gradient centrifugation revealed that only short DNA fragments were synthesized. There was no considerable fragmentation of template DNA during incubation of cdc8-1 strains under restrictive conditions.

An in vivo-in vitro alkaline DNA unwinding assay for hepatic DNA damage: Comparison with the alkaline sucrose gradient centrifugation technique

An in vivo-in vitro alkaline DNA unwinding assay for single-strand breaks and/or alkalilabile lesions in hepatic DNA is described. The assay involves isolation of hepatic nuclei from mice, alkaline denaturation and unwinding of hepatic DNA, separation of single- and double-stranded DNA by hydroxylapatite batch chromatography, and quantitation of DNA in chromatographic fractions by a fluorometric assay. The method allows for the sensitive detection of hepatic DNA damage following in vivo administration of xenobiotics. Using this procedure, DNA fragmentation was demonstrated in alkali after administration of as little as 0.5 mg/kg of N-nitrosodimethylamine (DMN) to male B6C3F1 mice. A comparison of this technique with a similar alkaline sucrose density gradient centrifugation assay demonstrated comparable limits of sensitivity for the two procedures.

The relationship between the antiviral activity of 5′-amino-5′-deoxythymidine and its incorporation into herpes simplex virus type 1

As part of our studies on the molecular basis for the antiherpes activity of 5′-AdThd (5′-amino-5′-deoxythymidine), a study of the HSV-1 DNA synthesized in infected Vero cells exposed to 5′-AdThd was undertaken. Unlike many other antiviral nucleoside analogs, 5′-AdThd did not inhibit HSV-1 DNA synthesis. Analysis of the DNA synthesized in the presence of [ 14C]5′-AdThd revealed that the analog was incorporated into the viral DNA in a dose-dependent manner and that the degree of incorporation correlated with the antiviral activity as measured by yield reduction assays. Analysis of the 5′-AdThd substituted DNA by centrifugation in neutral and alkaline sucrose gradients revealed no double-stranded breaks but an increase in single-stranded breaks, at very high concentrations of the analog. Analysis of HSV-1-specific RNAs revealed a shift from poly(A +) to poly(A -) RNA. The degree of this shift paralleled the substitution of 5′-AdThd for thymidine in the HSV-1 DNA.

Molecular dosimetry of DNA damage caused by alkylation II. The induction and repair of different classes of single-strand breaks in cultured mammalian cells treated with ethylating agents

Cultured Chinese hamster ovary cells were treated with ethylating agents. DNA lesions giving rise to single-strand breaks (SSB) or alkali-labile sites were measured by elution through membrane filters at pH 12.0 and pH 12.6, and by centrifugation in alkaline sucrose gradients after 1 h and 21 h lysis in alkali. Two agents with different tendencies to ethylate preferentially either at N or O atoms were compared, namely N-ethyl- N′-nitro- N-nitrosoguanidine (ENNG) and diethyl sulphate (DES). The compounds differed greatly in their potency to induce lesions, but the ratios of SSB, measured with different methods after a treatment for 30 min, did not differ significantly. This suggested that the spectrum of lesions induced by the two compounds is very similar. However, when both agents were studied with alkaline elution at pH 12.0 after a short treatment time (5 min) only ENNG was found to induce rapidly-repairable SSB. Most of these were rejoined already within 5 min after treatment. These results suggest that rapidly-repairable lesions occurring in DNA after treatment of mammalian cells with ethylating agents are due mainly to alkylation at O-atoms.

DNA damage in HeLa S3 cells by an antitumor drug ledakrin and other antitumor 1-nitro-9-aminoacridines

Ledakrin and seven other antitumor and cytotoxic derivatives of 1-nitro-9-aminoacridine were shown to induce DNA-single strand breaks in HeLa S3 cells as found by alkaline sucrose gradient centrifugation. The induced DNA damage is of non-random character. Some of Ledakrin-induced DNA breaks are probably generated by endonucleolytic cleavage in the course of repair processes as indicated by experiments with Novobiocin, an antibiotic preventing the incision step of DNA repair. Other Ledakrin-induced DNA breaks observed on alkaline sucrose gradients may arise from alkali-labile sites in DNA. Most of such sites seem to be converted to breaks after brief exposure to alkali. The extent of DNA damage by 1-nitro-9-aminoacridines was found to be correlated with cytotoxic activities of these compounds against HeLa S3 cells. Furthermore, Ledakrin and other derivatives seem to induce DNA-repair synthesis in HeLa S3 cells as judged by the stimulation of hydroxyurea (HU)-resistant incorporation of [ 3H] thymidine into DNA. The agents studied differ in their concentrations required to produce a considerable stimulation of DNA repair, whereas the maximal level of this effect is similar for all the derivatives assayed. The former values are correlated with cytotoxic activites of these compounds and seem to reflect the overall extent of DNA damage by 1-nitro-9-aminoacridines. Stimulation of DNA-repair synthesis is gradually shut off during prolonged incubation of the cells with Ledakrin or during postincubation of the cells in a drug-free medium. Such postincubation results also in the gradual accumulation of DNA-single strand breaks as observed by alkaline sucrose centrifugation. Hence, HeLa S3 cells are incapable of efficiently removing DNA damage by 1-nitro-9-aminoacridines, though the drug's action activates temporarily some repair mechanisms. The reported results suggest that overall DNA damage may contribute to the cytotoxic effects of 1-nitro-9-aminoacridines besides previously found ability of these agents to form interstrand DNA cross-links.