Split-dose recovery and protein synthesis in X-irradiated CHO cells

A temperature-sensitive mutant for protein synthesis, CHO-tsH1, has been compared to the wild-type (WT) cell, CHO-SC1, in single- and split-radiation-dose schemes. When the exponentially growing mutant and the wild-type cells were treated at 40 degrees C for up to 3 h prior to split doses of X rays, survival was progressively reduced in the mutant compared to the wild type. In addition, if a 2-h split-dose scheme was used with a treatment of 40 degrees C given before the first dose, between the dose fractions and after the second dose, the recovery from sublethal damage (SLD) was almost completely inhibited in the mutant cells. These observations implied that a pool of proteins was involved in the recovery from sublethal X-ray damage. However, if molecular repair was measured in the mutant cell by the alkaline-unwinding technique under the same time and temperature schemes as those demonstrating a reduction in the recovery from SLD, no difference in the kinetics of DNA strand rejoining was observed compared to similar measurements made under conditions permitting SLD recovery. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. Split-dose experiments were also done using cycloheximide to chemically inhibit protein synthesis. Under conditions which mimicked those used in the temperature-shift experiments both cell lines showed a reduction in the recovery from sublethal damage comparable in magnitude to that observed in the mutant cells when they were treated with 40 degrees C. Both the chemical and thermal inhibition of protein synthesis substantiate its necessity for the recovery from sublethal damage.     

Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase

Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death.     

TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1

The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3'- or 5'-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase 'poisons'. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3'-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3'-tyrosyl DNA phosphodiesterase (3'-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting Tdp1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of Tdp1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both Tdp1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in Tdp1(-)(/)(-) cells, suggesting that Tdp2 contributes to cellular 3'-TDP activity in the absence of Tdp1. Consistent with this idea, over-expression of human TDP2 in Tdp1(-)(/)(-)/Tdp2(-)(/)(-)(/)(-) DT40 cells increases DNA strand break repair rates and cell survival above that observed in Tdp1(-)(/)(-) DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of Tdp1. Finally, mice lacking both Tdp1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking Tdp1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of Tdp1. In contrast, we failed to detect a contribution for Tdp1 to repair Top2-mediated damage. Together, our data suggest that Tdp1 and Tdp2 fulfil overlapping roles following Top1-induced DNA damage, but not following Top2-induced DNA damage, in vivo.     

Hyperthermia, polyamine depletion, and inhibition of X-ray-induced DNA strand break repair

We have recently demonstrated that HeLa cells that had been depleted of polyamines by treatment with inhibitors of polyamine biosynthesis were deficient in their ability to repair X-ray-induced DNA strand breaks. Since it had previously been demonstrated that hyperthermic shock also inhibited strand break repair following X irradiation and that hyperthermia resulted in a leakage of polyamines from cells, it seemed of interest to examine whether the inhibition of repair by hyperthermia was related to this loss of cellular polyamines. In the present paper it is demonstrated that both polyamine depletion and hyperthermia inhibit strand closure, and that a combined treatment further reduces the rate of repair. In cells not depleted of polyamines, repair is restored to normal levels if hyperthermia treatment is followed by a 4-h incubation at 37 degrees C before X irradiation. In polyamine-depleted cells, this 37 degrees C incubation does not result in a return of repair ability. Polyamine supplementation was not effective in reversing hyperthermia-dependent repair inhibition, and, in fact, restoration of repair in control cells following hyperthermic shock corresponded to a time at which polyamines show a maximum decrease in those cells. These results suggest that the inhibition of repair and the increased radiosensitivity observed in hyperthermically treated cells is not related to polyamine depletion. However, data further suggest that polyamine-depleted cells may have other alterations, perhaps in chromatin, which render them more sensitive to thermal inhibition of repair.     

ATP hydrolysis by mammalian RAD51 has a key role during homology-directed DNA repair

Disruption of the gene encoding RAD51, the protein that catalyzes strand exchange during homologous recombination, leads to the accumulation of chromosome breaks and lethality in vertebrate cells. As RAD51 is implicated in BRCA1- and BRCA2-mediated tumor suppression as well as cellular viability, we have begun a functional analysis of a defined RAD51 mutation in mammalian cells. By using a dominant negative approach, we generated a mouse embryonic stem cell line that expresses an ATP hydrolysis-defective RAD51 protein, hRAD51-K133R, at comparable levels to the endogenous wild-type RAD51 protein, whose expression is retained in these cells. We found that these cells have increased sensitivity to the DNA-damaging agents mitomycin C and ionizing radiation and also exhibit a decreased rate of spontaneous sister-chromatid exchange. By using a reporter for the repair of a single chromosomal double-strand break, we also found that expression of the hRAD51-K133R protein specifically inhibits homology-directed double-strand break repair. Furthermore, expression of a BRC repeat from BRCA2, a peptide inhibitor of an early step necessary for strand exchange, exacerbates the inhibition of homology-directed repair in the hRAD51-K133R expressing cell line. Thus, ATP hydrolysis by RAD51 has a key role in various types of DNA repair in mammalian cells.     

Modified cellular metabolism in hybridomas subjected to hydrodynamic and other stresses

The responses of hybridoma growth and metabolism to hydrodynamic forces and changes in culture temperature have been examined. DNA synthesis is inhibited under conditions of intense hydrodynamic stress; however, cellular metabolic activity is increased perhaps to feed repair mechanisms. Sub-optimal temperature and nutrient deprivation increase cellular susceptibility to hydrodynamic stress, possibly through the inhibition of repair mechanisms. The reduced cell number in conditions of high agitation may be due at least in part to growth inhibition.     

Performance of seminal and nodal roots of wheat in stagnant solution: K+ and P uptake and effects of increasing O2 partial pressures around the shoot on nodal root elongation

Roots of intact wheat plants were grown for 7-12 d in stagnant nutrient solution, containing 0.1% agar, to mimic the lack of convection in waterlogged soil. Net K+ and P uptakes by seminal and nodal roots were measured separately using a split root system. For seminal roots in stagnant solution, net uptakes as a percentage of aerated roots were between 0% and 16% for P, while K+ ranged between 15% uptake and 54% loss. For the more waterlogging-tolerant nodal roots, net uptakes in stagnant nutrient solution, as a percentage of aerated roots, were 31-73% for P and 69-115% for K+. Elongation rates of nodal roots in stagnant nutrient were about 35-43% of those for roots in aerated solution. This partial inhibition occurred in these nodal roots despite their 15% porosity (v/v). Elevation of O2 partial pressures around the shoots to 40 kPa and then to 80 kPa substantially accelerated nodal root elongation in stagnant solution, demonstrating that most of the inhibition seen with ambient O2 around the shoots was associated with a restricted O2 supply to these nodal roots. Thus, in wheat nodal roots, with a partial pressure of 20 kPa O2 around the shoots, O2 diffusion from the shoots did not completely relieve the restrictions on elongation resulting from stagnancy in the nutrient solution. These results contrast with those in the literature for rice, in which roots function efficiently in stagnant solutions (0.1% agar). So, when wheat roots are aerenchymatous there are still restrictions to O2 diffusion in the gas space continuum between the atmosphere and the functional tissues of the roots. This poor acclimation must have been due to inefficiency of the aerenchymatous axes, which may include persistence of anoxic steles, and/or restricted O2 diffusion in other parts of the gas space continuum, in either the shoots and shoot-root junction or in the root tip.     

Hyperthermia inhibits homologous recombination repair and sensitizes cells to ionizing radiation in a time‐ and temperature‐dependent manner

Hyperthermia has long been known as a radio‐sensitizing agent that displays anti‐tumor effects, and has been developed as a therapeutic application. The mechanisms of hyperthermia‐induced radio‐sensitization are highly associated with inhibition of DNA repair. Our investigations aimed to show how hyperthermia inactivate homologous recombination repair in the process of sensitizing cells to ionizing radiation by using a series of DNA repair deficient Chinese Hamster cells. Significant differences in cellular toxicity attributable to hyperthermia at and above 42.5°C were observed. In wild‐type and non‐homologous end joining repair mutants, cells in late S phase showed double the amount heat‐induced radio‐sensitization effects of G1‐phase cells. Both radiation‐induced DNA double strand breaks and chromatin damage resulting from hyperthermia exposure was measured to be approximately two times higher in G2‐phase cells than G0/G1 cells. Additionally, G2‐phase cells took approximately two times as long to repair DNA damage over time than G0/G1‐phase cells. To supplement these findings, radiation‐induced Rad51 foci formations at DNA double strand break sites were observed to gradually dissociate in response to the temperature and time of hyperthermia exposure. Dissociated Rad51 proteins subsequently re‐formed foci at damage sites with time, and occurred in a trend also related to temperature and time of hyperthermia exposure. These findings suggest Rad51's dissociation and subsequent reformation at DNA double strand break sites in response to varying hyperthermia conditions plays an important role in hyperthermia‐induced radio‐sensitization. J. Cell. Physiol. 228: 1473–1481, 2013. © 2012 Wiley Periodicals, Inc.     

Changes in poly(ADP-ribose) level modulate the kinetics of DNA strand break rejoining

ADP-ribose polymers are rapidly synthesized in cell nuclei by the poly(ADP-ribose) polymerases PARP-1 and PARP-2 in response to DNA strand interruptions, using NAD(+) as precursor. The level of induced poly(ADP-ribose) formation is proportional to the level of DNA damage and can be decreased by NAD(+) or PARP deficiency, followed by poor DNA repair and genomic instability. Here we studied the correlation between poly(ADP-ribose) level and DNA strand break repair in lymphoblastoid Raji cells. Poly(ADP-ribose) synthesis was induced by 100 microM H(2)O(2) and intensified by the 1,4-dihydropyridine derivative AV-153. The level of poly(ADP-ribose) in individual cells was analyzed by quantitative in situ immunofluorescence and confirmed in whole-cell extracts by Western blotting, and DNA damage was assessed by alkaline comet assays. Cells showed a approximately 100-fold increase in poly(ADP-ribose) formation during the first 5 min of recovery from H(2)O(2) treatment, followed by a gradual decrease up to 15 min. This synthesis was completely inhibited by the PARP inhibitor NU1025 (100 microM) while the cells treated with AV-153, at non-genotoxic concentrations of 1 nM-10 microM, showed a concentration-dependent increase of poly(ADP-ribose) level up to 130% after the first minute of recovery. The transient increase in poly(ADP-ribose) level was strongly correlated with the speed and efficiency of DNA strand break rejoining (correlation coefficient r > or = 0.92, p<0.05). These results are consistent with the idea that poly(ADP-ribose) formation immediately after genome damage reflects rapid assembly and efficient functioning of repair machinery.     

Extensive and equivalent repair in both radiation-resistant and radiation-sensitive E. coli determined by a DNA-unwinding technique.

The extent of strand breakage and repair in irradiated E. coli B/r and Bs-1 was studied using a DNA-unwinding technique in denaturing conditions of weak alkali. Although these two strains show widely different responses to the lethal effects of ionizing radiation, they both have an equal capacity to repair radiation-induced breaks in DNA. Oxygen enhancement ratios for the killing of B/r and Bs-1 were respectively 4 and 2; but after repair in non-nutrient or nutrient post-irradiation conditions, the oxygen enhancement values for the residual strand breaks were always the same for the two strains. The equal abilities of E. coli B/r and E. coli Bs-1 to remove the strand breaks measured by this weak-alkali technique leads us to suggest that some other type of damage to either DNA or another macromolecule may play a major role in determining whether or not the cells survive to proliferate.     

Poly(ADP-ribose) and the response of cells to ionizing radiation

The activity of poly(ADP-ribose) polymerase is stimulated by DNA damage resulting from treatment of cells with ionizing radiation, as well as with DNA-damaging chemicals. The elevated polymerase activity can be observed at doses lower than those necessary for measurable reduction in cellular NAD concentration (less than 20 Gy). Several nuclear proteins, including the polymerase itself, are poly(ADP-ribosylated) at elevated levels in irradiated Chinese hamster cells. The addition of inhibitors of poly(ADP-ribose) polymerase to irradiated cells has been found to sensitize the cells to the lethal effects of the radiation, to inhibit the repair of potentially lethal damage, and to delay DNA strand break rejoining. Because of the nonspecificity of the inhibitors, however, it is as yet unknown whether their effects are directly related to the inhibition of poly(ADP-ribose) polymerase, to interference with the poly(ADP-ribosylation) of one or more chromosomal proteins, or to effects unrelated to the poly(ADP-ribosylation) process. The data are consistent with the involvement of poly(ADP-ribose) in the repair of radiation damage, but the nature of this involvement remains to be elucidated.     

APLF (C2orf13) is a novel human protein involved in the cellular response to chromosomal DNA strand breaks

Aprataxin and polynucleotide kinase (PNK) are DNA end processing factors that are recruited into the DNA single- and double-strand break repair machinery through phosphorylation-specific interactions with XRCC1 and XRCC4, respectively. These interactions are mediated through a divergent class of forkhead-associated (FHA) domain that binds to peptide sequences in XRCC1 and XRCC4 that are phosphorylated by casein kinase 2 (CK2). Here, we identify the product of the uncharacterized open reading frame C2orf13 as a novel member of this FHA domain family of proteins and we denote this protein APLF (aprataxin- and PNK-like factor). We show that APLF interacts with XRCC1 in vivo and in vitro in a manner that is stimulated by CK2. Yeast two-hybrid analyses suggest that APLF also interacts with the double-strand break repair proteins XRCC4 and XRCC5 (Ku86). We also show that endogenous and yellow fluorescent protein-tagged APLF accumulates at sites of H(2)O(2) or UVA laser-induced chromosomal DNA damage and that this is achieved through at least two mechanisms: one that requires the FHA domain-mediated interaction with XRCC1 and a second that is independent of XRCC1 but requires a novel type of zinc finger motif located at the C terminus of APLF. Finally, we demonstrate that APLF is phosphorylated in a DNA damage- and ATM-dependent manner and that the depletion of APLF from noncycling human SH-SY5Y neuroblastoma cells reduces rates of chromosomal DNA strand break repair following ionizing radiation. These data identify APLF as a novel component of the cellular response to DNA strand breaks in human cells.     

Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations

Utilizing the concept of synthetic lethality has provided new opportunities for the development of targeted therapies, by allowing the targeting of loss of function genetic aberrations. In cancer cells with BRCA1 or BRCA2 loss of function, which harbor deficiency of DNA repair by homologous recombination, inhibition of PARP1 enzymatic activity leads to an accumulation of single strand breaks that are converted to double strand breaks but cannot be repaired by homologous recombination. Inhibition of PARP has therefore been advanced as a novel targeted therapy for cancers harboring BRCA1/2 mutations. Preclinical and preliminary clinical evidence, however, suggests a potentially broader scope for PARP inhibitors. Loss of function of various proteins involved in double strand break repair other than BRCA1/2 has been suggested to be synthetically lethal with PARP inhibition. Inactivation of these genes has been reported in a subset of human cancers and might therefore constitute predictive biomarkers for PARP inhibition. Here we discuss the evidence that the clinical use of PARP inhibition may be broader than targeting of cancers in BRCA1/2 germ-line mutation carriers.Key words: homologous recombination, PARP inhibitor, BRCA1, BRCA2, PTEN, PALB2, EMSY, double strand break repair     

A Stronger DNA Damage-Induced G2 Checkpoint Due to Over-Activated CHK1 in the Absence of PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in multi-pathways to respond to DNA damage. Lack of or inhibition of PARP-1 activity leads to slow progress of cell cycle and sensitization of cells to different stresses. Recently, it was reported that besides the Ku- dependent main non-homologous end joining (NHEJ) pathway, there is a PARP-1-dependent complementary NHEJ pathway to repair DNA double strand break (DSB). Here we show that compared with PARP-1+/+ cells, PARP-1-/- cells display a much stronger G2 checkpoint response following ionizing radiation (IR). Treatment with Chk1 siRNA abolishes the stronger G2 checkpoint response and sensitizes PARP-1-/- cells to IR. These data indicate that the stronger G2 checkpoint response in PARP-1-/- cells is CHK1-dependent, which protects cells from IR-induced killing. We also show that 4-Amino-1,8-naphthalimide (4-AN, inhibitor of PARP) but not methoxyamine (inhibitor of base excision repair (BER)), affects IR-induced G2 arrest and cell sensitivity in PARP-1+/+ cells, resulting in the phenotypes similar to those of PARP-1-/- cells. These results indicate that DSB repair from the complementary NHEJ pathway of PARP-1, but not single strand break (SSB) repair from the BER function of PARP-1, may play an essential role in the over-activated CHK1 regulated G2 checkpoint response and radiosensitivity in PARP-1-/- cells.     

Inhibition of poly(ADP-ribose) polymerase causes increased DNA strand breaks without decreasing strand rejoining in alkylated HeLa cells

Treatment of alkylated HeLa cells with 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, increased the number of DNA strand breaks but did not affect the rate of strand rejoining. This suggests that an increase in DNA incision, not a decrease in ligation, results from the inhibition ofpoly(ADP-ribose) polymerase in cells recovering from DNA damaged by alkylating agents. Poly(ADP-ribose) DNA strand break DNA repair     

Chemical radiosensitization by misonidazole: production and repair of DNA single-strand breaks in Yoshida ascites tumor cells.

Formation of strand-breaks in DNA and its repair in Yoshida ascites tumor cells exposed to gamma radiation (100-400 Gy) in presence and absence of misonidazole (10 mM) were studied. The methodology involved pre-labelling of cellular DNA by 3H-thymidine during cell proliferation in rats, irradiation of cells in vitro and analysing sedimentation profile of DNA by ultracentrifugation in alkaline sucrose density gradients. Irradiation under euoxic conditions resulted in formation of about 1.5 times greater number of strand breaks as compared to those formed during irradiation under hypoxic conditions. Misonidazole (10 mM) by its presence along with the cells during irradiation under hypoxic conditions caused a 3-fold increase in the number of single strand breaks, but under euoxic conditions of irradiation the presence of misonidazole did not enhance the strand break formation. Incubation of cells irradiated in absence of misonidazole for 1 hr in tissue culture medium at 37 degrees C resulted in repair of substantial fraction of the strand breaks while there was no repair of the DNA strand breaks in cells irradiated in the presence of the chemical.     

Deficient repair of the transcribed strand of active genes in Cockayne's syndrome cells.

Removal of ultraviolet light induced cyclobutane pyrimidine dimers (CPD) from active and inactive genes was analyzed in cells derived from patients suffering from the hereditary disease Cockayne's syndrome (CS) using strand specific probes. The results indicate that the defect in CS cells affects two levels of repair of lesions in active genes. Firstly, CS cells are deficient in selective repair of the transcribed strand of active genes. In these cells the rate and efficiency of repair of CPD are equal for the transcribed and the nontranscribed strand of the active ADA and DHFR genes. In normal cells on the other hand, the transcribed strand of these genes is repaired faster than the nontranscribed strand. However, the nontranscribed strand is still repaired more efficiently than the inactive 754 gene and the gene coding for coagulation factor IX. Secondly, the repair level of active genes in CS cells exceeds that of inactive loci but is slower than the nontranscribed strand of active genes in normal cells. Our results support the model that CS cells lack a factor which is involved in targeting repair enzymes specifically towards DNA damage located in (potentially) active DNA.     

Genetic-dependent alterations in adrenal stress response and adrenocortical cell function of the domestic fowl (Gallus domesticus)

Strain-dependent differences in adrenocortical function were investigated in male White Leghorn domestic fowl. Adrenocortical function of Cornell K strain (K) (genetic control), autosomal dwarf strain (ADW), and sex-linked recessive dwarf strain (SLD) was evaluated in vivo by measuring plasma corticosterone and in vitro by measuring acute (2 hr) corticosterone production by enriched adrenocortical cell populations. Regardless of strain, there was an age-dependent decrease (27-57%) in plasma corticosterone from 1 to 12 weeks of age. However, there was a tendency for plasma corticosterone values of ADW and SLD to be, respectively, greater and less than that of K. In addition, at 12 weeks of age, plasma corticosterone responses of ADW and SLD to transient heat stress (50 degrees C, 30 min) were, respectively, 22.8% greater and 15.9% less than that of K. Strain differences in adrenal weight and relative adrenal weight (mg% body wt) were also apparent. At 12 weeks of age, adrenal weights of ADW and SLD were, respectively, 33 and 42% less than that of K, whereas relative adrenal weights were, respectively, 27.6% greater and 22.4% less than that of K. In addition there were strain-dependent differences in adrenocortical function at the cellular level. Although there were no consistent strain differences in basal and maximal corticosterone production by cells, there were strain differences in cellular sensitivity to ACTH and pregnenolone. On an equal cell concentration basis, the half-maximal steroidogenic concentrations (ED50 values or effective doses for 50% maximal effect) of ACTH for ADW and SLD adrenocortical cells were, respectively, 0.23 and 2.07 times the ED50 value for K cells. In addition, the ED50 value of pregnenolone for ADW cells was 0.46 times that for K and SLD cells. Since ED50 values are a measure of cellular sensitivity (the greater the ED50 value the lesser the cellular sensitivity), the order of sensitivity to ACTH was ADW greater than K greater than SLD and the order of sensitivity to pregnenolone was ADW greater than K = SLD. However, there were no strain differences in ED50 values of 8-bromo-cyclic AMP. These data suggest that strain differences in plasma corticosterone response to stress are, in part, due to differences in relative adrenal weight and differences in adrenocortical cell function.     

Defective repair of DNA single- and double-strand breaks in the bleomycin- and X-ray-sensitive Chinese hamster ovary cell mutant, BLM-2

Using filter elution techniques, we have measured the level of induced single- and double-strand DNA breaks and the rate of strand break rejoining following exposure of two Chinese hamster ovary (CHO) cell mutants to bleomycin or neocarzinostatin. These mutants, designated BLM-1 and BLM-2, were isolated on the basis of hypersensitivity to bleomycin and are cross-sensitive to a range of other free radical-generating agents, but exhibit enhanced resistance to neocarzinostatin. A 1-h exposure to equimolar doses of bleomycin induces a similar level of DNA strand breaks in parental CHO-K1 and mutant BLM-1 cells, but a consistently higher level is accumulated by BLM-2 cells. The rate of rejoining of bleomycin-induced single- and double-strand DNA breaks is slower in BLM-2 cells than in CHO-K1 cells. BLM-1 cells show normal strand break repair kinetics. The level of single- and double-strand breaks induced by neocarzinostatin is lower in both BLM-1 and BLM-2 cells than in CHO-K1 cells. The rate of repair of neocarzinostatin-induced strand breaks is normal in BLM-1 cells but retarded somewhat in BLM-2 cells. Thus, there is a correlation between the level of drug-induced DNA damage in BLM-2 cells and the bleomycin-sensitive, neocarzinostatin resistant phenotype of this mutant. Strand breaks induced by both of these agents are also repaired with reduced efficiency by BLM-2 cells. The neocarzinostatin resistance of BLM-1 cells appears to be a consequence of a reduced accumulation of DNA damage. However, the bleomycin-sensitive phenotype of BLM-1 cells does not apparently correlate with any alteration in DNA strand break induction or repair, as analysed by filter elution techniques, suggesting an alternative mechanism of cell killing.