UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages

UVA (320–400 nm) represents the main spectral component of solar UV radiation, induces pre-mutagenic DNA lesions and is classified as Class I carcinogen. Recently, discussion arose whether UVA induces DNA double-strand breaks (dsbs). Only few reports link the induction of dsbs to UVA exposure and the underlying mechanisms are poorly understood. Using the Comet-assay and γH2AX as markers for dsb formation, we demonstrate the dose-dependent dsb induction by UVA in G₁-synchronized human keratinocytes (HaCaT) and primary human skin fibroblasts. The number of γH2AX foci increases when a UVA dose is applied in fractions (split dose), with a 2-h recovery period between fractions. The presence of the anti-oxidant Naringin reduces dsb formation significantly. Using an FPG-modified Comet-assay as well as warm and cold repair incubation, we show that dsbs arise partially during repair of bi-stranded, oxidative, clustered DNA lesions. We also demonstrate that on stretched chromatin fibres, 8-oxo-G and abasic sites occur in clusters. This suggests a replication-independent formation of UVA-induced dsbs through clustered single-strand breaks via locally generated reactive oxygen species. Since UVA is the main component of solar UV exposure and is used for artificial UV exposure, our results shine new light on the aetiology of skin cancer.     

The formation and repair of single-strand breaks in DNA of cultured mammalian cells treated with UV-light, methylating agents or 4-nitroquinoline-1-oxide.

The technique of sedimentation in alkaline sucrose was used to examine the formation and repair of single-strand (SS) breaks in cultured mammalian cells that were treated with methyl methanesulfonate (MMS), methyl nitrosourea (MNUA), 4-nitroquinoline-1-oxide (4NQO) or UV-light. The SS breaks induced by MMS and 4NQO were largely repaired by HeLa cells during a 5-h post-treatment incubation. The SS breaks induced by MNUA and UV-light were not repaired by HeLa cells. L-cells were not able to repair the SS breaks induced by any of the agents, which correlates with the deficiency of these cells for repair synthesis of DNA. The following conclusions are discussed. MNUA and UV-light produce modifications in DNA which are not repaired but are translated into SS breaks in alkali. MMS produces SS breaks intracellularly but these are not derived from a simple depurination of methylated purines. 4NQO produces a modification in DNA which is translated into an SS break in alkali but which can be removed by an intracellular process.     

Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells

DNA-dependent protein kinase (DNA-PK) is activated in a two-step process whereby the Ku heterodimer first binds to the DNA double-strand breaks (dsbs) and then the DNA-PK catalytic subunit (cs) is recruited to form a repair complex. Oxidative stress is simultaneously generated along with DNA damage by ionizing radiation or chemotherapeutic agents whose impact on the DNA-PK activity has not previously been investigated. Here we show that the DNA damage-induced kinase activity of DNA-PK was modulated by oxidative stress, which was induced along with DNA dsbs in chlorambucil (Cbl)-exposed cells. Pretreatment with the antioxidants, 2(3)-t-butyl-4-hydroxyanisole or N-acetyl-l-cysteine enhanced the amount of DNA-PKcs phosphorylated at threonine 2609 (DNA-PK^pThr2609) at the DNA dsbs and DNA-PK activity. Conversely, oxidative stress induced by l-buthionine (SR)-sulfoximine or glucose oxidase decreased the DNA-PK activity in Cbl-exposed cells. In addition, DNA-PK^pThr2609 was poorly detectable at the site of DNA dsbs, as shown by colocalization to DNA-end-binding pH2AX or p53BP1. There was no change in the protein levels of DNA-PKcs, Ku70, or Ku86. Data from these studies provide the first evidence that oxidative stress effects posttranslational modification and assembly of DNA-PK complex at DNA dsbs, and thereby repair of DNA dsbs.     

Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells

DNA-dependent protein kinase (DNA-PK) is activated in a two-step process whereby the Ku heterodimer first binds to the DNA double-strand breaks (dsbs) and then the DNA-PK catalytic subunit (cs) is recruited to form a repair complex. Oxidative stress is simultaneously generated along with DNA damage by ionizing radiation or chemotherapeutic agents whose impact on the DNA-PK activity has not previously been investigated. Here we show that the DNA damage-induced kinase activity of DNA-PK was modulated by oxidative stress, which was induced along with DNA dsbs in chlorambucil (Cbl)-exposed cells. Pretreatment with the antioxidants, 2(3)-t-butyl-4-hydroxyanisole or N-acetyl-l-cysteine enhanced the amount of DNA-PKcs phosphorylated at threonine 2609 (DNA-PK^pThr2609) at the DNA dsbs and DNA-PK activity. Conversely, oxidative stress induced by l-buthionine (SR)-sulfoximine or glucose oxidase decreased the DNA-PK activity in Cbl-exposed cells. In addition, DNA-PK^pThr2609 was poorly detectable at the site of DNA dsbs, as shown by colocalization to DNA-end-binding pH2AX or p53BP1. There was no change in the protein levels of DNA-PKcs, Ku70, or Ku86. Data from these studies provide the first evidence that oxidative stress effects posttranslational modification and assembly of DNA-PK complex at DNA dsbs, and thereby repair of DNA dsbs.     

DNA damage in human colonic mucosa cells induced by bleomycin and the protective action of vitamin E

Using the alkaline comet assay, we showed that bleomycin at 0.1-5 microg/ml induced DNA strand breaks and/or alkali-labile sites, measurable as the comet tail moment, in human colonic mucosa cells. This DNA damage was completely repaired during a 120-minute post-treatment incubation of the cells. Post-treatment of the bleomycin-damaged DNA with 3-methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing alkylated bases, gave rise to a significant increase in the extent of DNA damage, indicating that the drug could induce alkylative bases in DNA. We did not observe any change in the comet tail moment in the presence of catalase. Vitamin E ((+)-alpha -tocopherol) decreased DNA damage induced by bleomycin. The results obtained suggest that hydrogen peroxide might not be involved in the formation of DNA lesions induced by bleomycin in the colonic mucosa cells.     

Induction of DNA double-strand breaks in Chinese hamster V79 cells by 2-chlorodeoxyadenosine

2-Chlorodeoxyadenosine was found to induce DNA double-strand breaks as well as cell death in log-phase Chinese hamster V79 cells. The induction of DNA double-strand breaks, measured by a neutral elution technique, was observed after a 2-h incubation of the cells in the presence of 5 microM of 2-chlorodeoxyadenosine, but these breaks were almost rejoined by a subsequent 1-h incubation, even though this drug was present in the medium during incubation. This repair was prevented by the addition of nicotinamide, which is known to inhibit poly(ADP-ribose) synthesis that is strongly associated with the DNA ligation, but not prevented by the addition of 9-beta-D-arabinofuranosyladenine (araA), which is known to inhibit DNA polymerization. These results suggest that the repair of CdA-induced double-strand breaks is achieved by ligation alone without DNA polymerization. When 35 microM of cycloheximide and 1.3 mM of dibutyryl cAMP were added to the medium, it was found that the induction of double-strand breaks by 2-chlorodeoxyadenosine was suppressed, while the cytotoxicity of 2-chlorodeoxyadenosine measured by colony-forming ability was not interfered with. These results suggest that the induction of DNA double-strand breaks is not associated with the cytotoxicity of this drug.     

DNA replication and repair in ataxia telangiectasia cells exposed to bleomycin

A marked increase in sensitivity to bleomycin was observed in two ataxia telangiectasia (AT) lymphoblastoid cell lines compared to that in cell lines from two normal individuals. This sensitivity was obtained at two different concentrations of bleomycin. While normal cells showed a rapid recovery of ability to divide, there was no indication of such a recovery in AT cells up to 120 h after bleomycin treatment. A similar level of breakage of DNA occurred in both cell types after incubation with bleomycin. The rate of repair of these breaks was also the same. DNA synthesis was found to be more resistant to bleomycin in AT cells than in control cells. The latter data are in keeping with results previously obtained using ionizing radiation.     

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.     

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.     

DNA breaks and repair in the mouse leukemia L1210 cells exposed to three different types of interstrand DNA cross-linkers

DNA breaks and repair in mouse leukemia L1210 cells treated with 3 different types of cross-linkers, mitomycin C (MMC), 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride (ACNU) and SN-07 (a macromolecular antibiotic), were studied. Measured in D37 values, MMC gave the highest number of cross-links per lethal 'hit' directly after the 1-h treatment in the alkaline elution assay, followed by ACNU and SN-07. A good dose-response increase in induced interstrand DNA cross-linking frequency was observed in cells treated with 2.5-10 micrograms/ml MMC and with 10-100 micrograms/ml ACNU for 1 h with and without 24-h post-incubation. After 6-h post-incubation, the highest frequency of cross-linking was observed in cells treated with 2.5 micrograms/ml MMC and 30 micrograms/ml ACNU, while cross-link production continued in the cells treated with SN-07 for 12-h post-incubation. No significant increase in DNA breaks was observed in cells treated with MMC throughout 24-h post-incubation. The highest frequency of single-strand DNA breaks in cells treated with ACNU was observed immediately after the treatment and they disappeared after 6-h post-incubation. After 24-h post-incubation, a marked enhancement of the DNA breaks was observed in cells treated with SN-07 and the cells contained double-strand DNA breaks also. RNA synthesis was not affected in the cells treated with 10 micrograms/ml MMC and slightly inhibited to 70% of control in those treated with 100 micrograms/ml ACNU, while DNA synthesis in both cells was significantly inhibited after 24-h post-incubation. By contrast, both RNA and DNA synthesis were completely inhibited in cells treated with 8.0 micrograms/ml SN-07.     

Alzheimer's disease fibroblasts have normal repair of N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA damage determined by the alkaline elution technique

Cultured fibroblast strains from two normal persons and from two patients with the neurodegeneration of Alzheimer's disease were exposed to the alkylating chemical N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Immediately after exposure and also after a 24-h repair incubation period the single-strand breaks in the cells' DNA were quantified by the alkaline elution technique. In contrast to a report by others using alkaline elution, MNNG, and these same strains, we found no evidence of deficient repair of MNNG-induced DNA damage in the Alzheimer's disease cells. The putative DNA repair defect in Alzheimer's disease should be investigated by methods other than the alkaline elution technique which measures only a small fraction of the damage induced by an alkylating chemical such as MNNG.     

Degradation of DNA and structure-activity relationship between bleomycins A2 and B2 in the absence of DNA repair.

The contribution of DNA repair to the net number of DNA breaks produced during chemical degradation of DNA was determined by using temperature-sensitive mutant cells deficient in ATP-dependent DNA ligase [poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase, EC 6.5.1.1]. In a very sensitive assay for determining lesions introduced into Saccharomyces cerevisiae DNAs, 2-14C- and 6-3H-prelabeled DNAs from ligase-proficient and ligase-deficient cells were sedimented together through precalibrated, isokinetic alkaline sucrose gradients. DNA ligation was slower after chemical degradation of DNA by bleomycin than after gamma irradiation. DNA breaks increased approximately linearly with drug concentrations, and were approximately equivalent for ligase-proficient and ligase-deficient cells. These results were unexpected because ligase-deficient, but not ligase-proficient, cells lacked the capacity to eliminate DNA breaks produced by bleomycin. The results indicated that DNA repair did not occur during the chemical degradation of DNA under the experimental conditions. Bleomycin B2 produced considerably more DNA breaks than bleomycin A2 over a range of concentrations in ligase-proficient cells, which tolerated higher numbers of DNA breaks in general than ligase-deficient cells. The chemical analogues are structurally identical except for their cationic C-terminal amine. The actual number of DNA breaks produced by bleomycin A2 or bleomycin B2, and not the concentration of bleomycin A2 or bleomycin B2 per se, determined the amount of cell killing. DNA repair is critical in quantitating DNA breaks produced by chemicals, but was ruled out as a factor in the higher DNA breakage by bleomycin B2 than bleomycin A2.     

Biochemical evidence for two different mechanisms for bleomycin-induced cell killing.

Using pulsed-field gel electrophoresis, we have measured the ability of two bleomycin-sensitive mutants, XR-1 and BL-10, to repair DNA double-strands breaks (DSB). XR-1 was originally isolated by its hypersensitivity to killing with ionizing radiation, but we have also shown that it is sensitive to killing with bleomycin. In contrast, BL-10 was isolated by its extreme sensitivity to killing with bleomycin, and it is not cross-sensitive to other DNA breaking agents. A 1-h treatment of bleomycin induces a similar number of DNA double-strand breaks in XR-1, BL-10 and CHO cells. However, XR-1 is unable to repair bleomycin-induced DNA double-strand breaks, whereas BL-10 possesses the same kinetics of repair as parental CHO. These data lead us to conclude that at least two mechanisms of killing exist for bleomycin; one of them is DNA DSB-dependent, and the other seems to be DNA DSB-independent.     

Menage á trois: Double strand break repair,V(D)J recombination and DNA-PK

All organisms possess mechanisms to repair double strand breaks (dsbs) generated in their DNA by damaging agents. Site-specific dsbs are also introduced during V(D)J recombination. Four complementation groups of radiosensitive rodent mutants are defective in the repair of dsbs, and are unable to carry out V(D)J recombination effectively. The immune defect in Severe Combined Immunodeficient (scid) mice also results from an inability to undergo effective V(D)J recombination, and scid cell lines display a repair defect and belong to one of these complementation groups. These findings indicate a mechanistic overlap between the processes of DNA repair and V(D)J recombination. Recently, two of the genes defined by these complementation groups have been identified and shown to encode components of DNA-dependent protein kinase (DNA-PK). We review here the three fields which have become linked by these findings, and discuss the involvement of DNA-PK in dsb rejoining and in V(D)J recombination.     

Cellular and molecular effects of bleomycin are modulated by heat shock in Saccharomyces cerevisiae

To study some mechanisms underlying the stress responses in eukaryotic cells, we investigated the effect of heat shock (HS) on the induction of DNA double strand breaks as well as on potentially lethal and mutagenic events induced by the radiomimetic antibiotic bleomycin (BLM) in Saccharomyces cerevisiae. Haploid wild-type yeast cells in the logarithmic phase of growth were exposed to different concentrations of BLM (0-30 microg/ml, 1.5 h) without and with a previous HS (38 degrees C, 1 h). Immediately after treatments, survival as well as mutation frequency were determined, and quantitative analysis of chromosomal DNA by laser densitometry were performed both immediately after treatments and after incubation of cells during different time intervals in liquid nutrient medium free of BLM. Our results indicate that HS induces resistance to potentially lethal and mutagenic effects of BLM. Quantitative analysis of chromosomal DNA performed immediately after treatments showed the same DNA fragmentation, either upon BLM as single agent or preceded by HS. However, HS pretreated cells incubated during 4 h in liquid nutrient medium free of BLM repaired DNA double strand breaks more efficiently as compared to non-pretreated cells. On this basis, we propose that the observed HS-induced resistance to BLM depends on a regulatory network acting after DNA-induced damage, which includes genes involved in DNA repair, HS response and DNA metabolism.     

DNA signals for G2 checkpoint response in diploid human fibroblasts

DNA (deoxyribonucleic acid) signals that induce the G2 checkpoint response were examined using proliferative secondary cultures of diploid human fibroblasts. Treatments that generated DNA double-strand breaks (DSBs) directly were effective inducers of checkpoint response, generally producing >80% inhibition of mitosis (G2 delay) and the kinase activity of M-phase-promoting factor within 2 h of treatment. Effective inducers of G2 checkpoint response included γ-irradiation and the cancer chemotherapeutic drugs, bleomycin and etoposide. Treatments that produced DNA single-strand breaks, directly or indirectly through nucleotide excision repair, were not effective inducers of G2 delay. Ineffective treatments included incubation with camptothecin, an inhibitor of topoisomerase I (topo I), and irradiation with sublethal fluences of UVC, followed by incubation with aphidicolin. Transient severe inhibition of DNA synthesis with aphidicolin did not affect mitosis substantially, suggesting that the replication arrest input to the G2 checkpoint required more than brief inhibition of DNA synthesis. In contrast, moderate camptothecin-induced inhibition of DNA synthesis was associated with a strong inhibition of mitosis that developed 4–12 h after drug treatment. This result suggested that G2 delay was not expressed until the cells that were in S-phase at the time of treatment with camptothecin proceeded into G2. DNA damage was not necessary for induction of mitotic delay. An inhibitor of topoisomerase II (topo II), ICRF-193, which inhibits chromatid decatenation in G2 cells without damaging DNA, induced a severe inhibition of mitosis and M-phase-promoting factor kinase activity. The results suggest that DNA double-strand breaks and insufficiency of chromatid decatenation effectively induce the G2 checkpoint response, but DNA single-strand breaks do not.     

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.     

DBD-fish on neutral comets: simultaneous analysis of DNA single- and double-strand breaks in individual cells

Humanblood leukocytes exposed to X-rays were immersed in an agarose microgel on a slide, extensively deproteinized, and electrophoresed under neutral conditions. Following this single-cell gel electrophoresis assay, characteristics of DNA migration (i.e., area of the comet) are related to the DNA double-strand breaks (dsbs) yield. After electrophoresis, comets were briefly incubated in an alkaline unwinding solution, transforming DNA breaks and alkali-labile sites into restricted single-stranded DNA (ssDNA) motifs. These motifs behave as target sites for hybridization with a whole genome probe, following the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure. As DNA breakage increases with dose, more ssDNA is produced in the comet by the alkali and more DNA probe hybridizes, resulting in an increase in the mean fluorescence intensity. Since radiation-induced DNA single-strand breaks (ssbs) are far more frequent than dsbs, the mean fluorescence intensity of the DBD-FISH signal from the comet is related to the ssb level, whereas the surface area of the same comet signal is indicative of the dsb yield. Thus, both DNA break types may be simultaneously analyzed in the same cell. This was confirmed in a repair assay performing the DBD-FISH on neutral comets from a human cell line defective in the repair of dsbs. Otherwise, treatment with hydrogen peroxide, a main inducer of ssbs, increased the mean fluorescence intensity, but not the surface, of X-ray-exposed human leukocytes.     

125IdUrd-induced chromosome fragments, assayed by premature chromosome condensation, and DNA double-strand breaks have similar repair kinetics in G1-phase CHO-cells

The effect of 125I-decay on cell lethality, and induction of chromosome and DNA damage, was studied in synchronous non-cycling, G1-phase CHO-cells. For this purpose a population of mitotic cells was allowed to divide and progress through S-phase in the presence of 125IdUrd. Cells were subsequently transferred to conditioned medium (C-med) obtained from plateau-phase cultures that allowed cells to divide and accumulate in G1-phase in a non-cycling state. To accumulate 125I-induced damage, cells were kept frozen at -80 degrees C. Freezing was carried out using a new method that optimally preserves cell integrity. After various times of cold storage, cells were thawed and assayed for survival, DNA and chromosome damage, either immediately or after various times in C-med. Neutral filter elution was used to assay repair of DNA double-strand breaks (dsbs), and premature chromosome condensation was used to assay repair of chromosome fragments and induction of ring chromosomes. The results indicate very little repair at the cell survival level (repair of PLD). At the DNA level an efficient repair of DNA dsbs was observed, with kinetics similar to those observed after exposure to X-rays. At the chromosome level a fast repair of prematurely condensed chromosome fragment was observed, with a concomitant increase in the number of ring chromosomes induced. The repair kinetics of chromosome fragments and DNA dsbs were very similar, suggesting that DNA dsbs may underlie chromosome fragmentation.     

Detection and repair of a UV-induced photosensitive lesion in the DNA of human cells

Irradiation with UV light results in damage to the DNA of human cells. The most numerous lesions are pyrimidine dimers; however, other lesions are known to occur and may contribute to the overall deleterious effect of UV irradiation. We have observed evidence of a UV-induced lesion other than pyrimidine dimers in the DNA of human cells by measuring DNA strand breaks induced by irradiating with 313-nm light following UV (254-nm) irradiation. These breaks, measured by alkaline sucrose sedimentation, increased linearly with the dose of UV light over the range tested (10-40 J/m2). The breaks cannot be photolytically induced 5 h after a UV dose of 20 J/m2 in normal cells; however, in xeroderma pigmentosum variant cells, the breaks are inducible for up to 24 h after UV irradiation. Xeroderma pigmentosum group A cells in the same 5-h period show an increase in the number of strand breaks seen with 313-nm light photolysis from about 2 to 4 breaks/10(9) dalton DNA. These breaks can then be induced for up to 24 h. These data suggest that, in normal cells, the lesion responsible for this effect is rapidly repaired or altered; whereas, in xeroderma pigmentosum variant cells it seems to remain unchanged. Some change apparently occurs in the DNA of xeroderma pigmentosum group A cells which results in an increase in photolability. These data indicate a deficiency in DNA repair of xeroderma pigmentosum variant cells as well as in xeroderma pigmentosum group A cells.