The DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) Are stimulated by bulky adduct-containing DNA.

A variety of environmental, carcinogenic, and chemotherapeutic agents form bulky lesions on DNA that activate DNA damage checkpoint signaling pathways in human cells. To identify the mechanisms by which bulky DNA adducts induce damage signaling, we developed an in vitro assay using mammalian cell nuclear extract and plasmid DNA containing bulky adducts formed by N-acetoxy-2-acetylaminofluorene or benzo(a)pyrene diol epoxide. Using this cell-free system together with a variety of pharmacological, genetic, and biochemical approaches, we identified the DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) as bulky DNA damage-stimulated kinases that phosphorylate physiologically important residues on the checkpoint proteins p53, Chk1, and RPA. Consistent with these results, purified DNA-PK and ATM were directly stimulated by bulky adduct-containing DNA and preferentially associated with damaged DNA in vitro. Because the DNA damage response kinase ATM and Rad3-related (ATR) is also stimulated by bulky DNA adducts, we conclude that a common biochemical mechanism exists for activation of DNA-PK, ATM, and ATR by bulky adduct-containing DNA.

Comparison of clastogen-induced gene expression profiles in wild-type and DNA repair-deficient Rad54/Rad54B cells.

BACKGROUND: Previously we found that Rad54/Rad54B cells are more sensitive towards mitomycin C (MMC) as compared to wild-type (WT) cells. This difference in sensitivity was absent upon exposure to other clastogens like bleomycin (BLM) and gamma-radiation. In order to get further insight into possible underlying mechanisms, gene expression changes in WT and Rad54/Rad54B MEFs (mouse embryonic fibroblasts) after exposure to the clastogens MMC and BLM were investigated. Exposures of these cells to mutagens (N-ac-AAF and ENU) and vehicle were taken as controls. RESULTS: Most exposures resulted in an induction of DNA damage signaling and apoptosis genes and a reduced expression of cell division genes in cells of both genotypes. As expected, responses to N-ac-AAF were very similar in both genotypes. ENU exposure did not lead to significant gene expression changes in cells of both genotypes, presumably due to its short half-life. Gene expression responses to clastogens, however, showed a genotype-dependent effect for BLM and MMC. MMC treated Rad54/Rad54B MEFs showed no induction of p53-signaling, DNA damage response and apoptosis as seen for all the other treatments. CONCLUSION: These data support our finding that different types of clastogens exist and that responses to these types depend on the DNA repair status of the cells.

Developmental decline in DNA repair in neural retina cells of chick embryos. Persistent deficiency of repair competence in a cell line derived from late embryos.

Neural retinas of 6-day-old chick embryos synthesize DNA and are able to carry out DNA excision repair. However, in contrast to the situation in human cells, the maximum rate of repair induced by N-acetoxy acetylaminofluorene (AAAF) is no greater than that induced by methyl methanesulfonate (MMS). With advancing differentiation of the retina in the embryo, cell multiplication and DNA synthesis decline and cease, and concurrently the cells lose the ability to carry out DNA excision repair. Thus, in 15-16-day embryos, in which the level of DNA synthesis is very low, DNA repair is barely detectable. If retinas from 14-day embryos are dissociated with trypsin and the cell suspension is plated in growth- promoting medium, DNA synthesis is reinitiated; however, in these cultures there is no detectable repair of MMS-induced damage, and only low levels of repair are observed after treatment with AAAF. A cell line was produced, by repeated passaging of these cultures, in which the cell population reached a steady state of DNA replication. However, the cell population remained deficient in the ability to repair MMS-induced damage. This cell line most likely predominantly comprises cells of retino-glial origin. Possible correlations between deficiency in DNA repair mechanisms in replicating cells and carcinogenesis in neural tissues are discussed.

Involvement of mouse Rev3 in tolerance of endogenous and exogenous DNA damage.

The Rev3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta that is implicated in mutagenic translesion synthesis of damaged DNA. To investigate the function of its mouse homologue, we have generated mouse embryonic stem cells and mice carrying a targeted disruption of Rev3. Although some strain-dependent variation was observed, Rev3(-/-) embryos died around midgestation, displaying retarded growth in the absence of consistent developmental abnormalities. Rev3(-/-) cell lines could not be established, indicating a cell-autonomous requirement of Rev3 for long-term viability. Histochemical analysis of Rev3(-/-) embryos did not reveal aberrant replication or cellular proliferation but demonstrated massive apoptosis in all embryonic lineages. Although increased levels of p53 are detected in Rev3(-/-) embryos, the embryonic phenotype was not rescued by the absence of p53. A significant increase in double-stranded DNA breaks as well as chromatid and chromosome aberrations was observed in cells from Rev3(-/-) embryos. The inner cell mass of cultured Rev3(-/-) blastocysts dies of a delayed apoptotic response after exposure to a low dose of N-acetoxy-2-acetylaminofluorene. These combined data are compatible with a model in which, in the absence of polymerase zeta, double-stranded DNA breaks accumulate at sites of unreplicated DNA damage, eliciting a p53-independent apoptotic response. Together, these data are consistent with involvement of polymerase zeta in translesion synthesis of endogenously and exogenously induced DNA lesions.

Molecular characterization of an acidic region deletion mutant of Cockayne syndrome group B protein.

Cockayne syndrome (CS) is a human genetic disorder characterized by post-natal growth failure, neurological abnormalities and premature aging. CS cells exhibit high sensitivity to UV light, delayed RNA synthesis recovery after UV irradiation and defective transcription-coupled repair (TCR). Two genetic complementation groups of CS have been identified, designated CS-A and CS-B. The CSB gene encodes a helicase domain and a highly acidic region N-terminal to the helicase domain. This study describes the genetic characterization of a CSB mutant allele encoding a full deletion of the acidic region. We have tested its ability to complement the sensitivity of UV61, the hamster homolog of human CS-B cells, to UV and the genotoxic agent N-acetoxy-2-acetylaminofluorene (NA-AAF). Deleting 39 consecutive amino acids, of which approximately 60% are negatively charged, did not impact on the ability of the protein to complement the sensitive phenotype of UV61 cells to either UV or NA-AAF. Our data indicate that the highly acidic region of CSB is not essential for the TCR and general genome repair pathways of UV- and NA-AAF-induced DNA lesions.

Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro.

The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, global genome repair (GGR) and transcription-coupled repair (TCR). In contrast to somatic cells, little is known regarding the expression of these pathways in germ cells. To address this basic question, we have studied NER in rat spermatogenic cells in crude cell suspension, in enriched cell stages and within seminiferous tubules after exposure to UV or N-acetoxy-2-acetylaminofluorene. Surprisingly, repair in spermatogenic cells was inefficient in the genome overall and in transcriptionally active genes indicating non-functional GGR and TCR. In contrast, extracts from early/mid pachytene cells displayed dual incision activity in vitro as high as extracts from somatic cells, demonstrating that the proteins involved in incision are present and functional in premeiotic cells. However, incision activities of extracts from diplotene cells and round spermatids were low, indicating a stage-dependent expression of incision activity. We hypothesize that sequestering of NER proteins by mispaired regions in DNA involved in synapsis and recombination may underlie the lack of NER activity in premeiotic cells.

Nonspecific inhibition of DNA repair by promoting and nonpromoting phorbol esters.

The effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and its nonpromoting structural analogue, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (Me-TPA), on N-acetoxy-2-acetylaminofluorene-elicited DNA repair and replicative DNA synthesis was measured in normal human fibroblasts. Both esters inhibited DNA repair synthesis, and Me-TPA was nearly as effective as TPA. In addition, TPA inhibited replicative DNA synthesis. These findings showed that inhibition of DNA repair synthesis may not be a major factor in the mechanisms of action of tumor promoters.

Reduced capacity for DNA repair synthesis in patients with or genetically predisposed to colorectal cancer.

Peripheral resting mononuclear leukocytes were compared for their capacities to repair DNA lesions induced by a 1-hour exposure to a standardized 10-microM dose of N-acetoxy-N-2-fluorenylacetamide (N-AcO-2-FAA). Leukocytes from the following 3 groups were studied: 39 control subjects, 40 patients after colonic resection because of colorectal cancer (disease-free at the time of this study), and 28 individuals with a hereditary predisposition to colorectal cancer. Although the level of N-AcO-2-FAA that bound to mononuclear leukocyte DNA was the same for the various population groups, the level of N-AcO-2-FAA-induced unscheduled DNA synthesis (UDS) was significantly reduced in the mononuclear leukocytes of individuals who had had colorectal cancer or a genetic predisposition for the disease. These findings indicate that a deficiency in mononuclear leukocyte DNA repair synthesis is associated with the development of colorectal cancer in these populations. Our observation of this nonspecific UDS deficiency (relating to colorectal cancer) was not explained by experimental variations among the sampled groups with regard to individual differences in lymphocyte heterogeneity, age, sex, smoking habits, or blood pressure.

Clinical symptoms and DNA repair characteristics of xeroderma pigmentosum patients from Germany.

Sixty-one xeroderma pigmentosum (XP) patients living in the Federal Republic of Germany were investigated. Clinical symptoms were correlated with DNA repair parameters measured in fibroblasts grown from skin biopsies. Classification according to the international complementation groups revealed that of the 61 patients 3 belonged to group A, 26 to group C, 16 to group D, 3 to group E, and 2 to group F; 11 were of the XP variant type. A striking clinical aspect was the frequency of histogenetically different skin tumors varying from one XP complementation group to the other: squamous and basal cell carcinomas predominated in XP group C; lentigo maligna melanomas were most frequent in group D; basal cell carcinomas occurred preferentially in group E and XP variants. Three DNA repair parameters were determined for 46 fibroblast strains: colony-forming ability (D0); DNA repair synthesis (G0); and DNA-incising capacity (E0). Dose-response experiments with up to 13 dose levels were performed throughout to achieve sufficient experimental accuracy. DNA-damaging treatments included UV light, the "UV-like" carcinogen N-acetoxy-2-acetylaminofluorene, and the alkylating carcinogens methyl methanesulfonate and N-methyl-N-nitrosourea. Comparison of clinical signs and repair data was made on the basis of D0, G0, and E0 values of both individual cell strains and weighted means of XP complementation groups. Despite considerable clinical and biochemical heterogeneity within complementation groups distinctive features emerged. In general, D0, G0, and E0 values of all XP strains investigated, including XP variants, were found to be reduced upon treatment with UV light or N-acetoxy-2-acetylaminofluorene. After treatment with UV light or N-acetoxy-2-acetylaminofluorene, cell strains in which DNA-incising capacity was reduced also showed a similar reduction in both colony-forming ability and DNA repair synthesis. Consequently, the weighted mean D0, G0, and E0 values of XP complementation groups and XP variants correlated with each other. Furthermore, the onset of both early dermatological symptoms of XP and tumor growth correlated with the extent of DNA repair defects. Of 45 XP fibroblast strains checked for colony-forming ability after treatment with methyl methanesulfonate only 3 cell strains from group D were found to be more sensitive than normal controls, suggesting that overall repair in XP strains was equal to that in controls. Weighted means of DNA repair synthesis of XP complementation groups, however, showed reductions hinting at impaired excision of distinct alkylated bases.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of depurination in mutagenesis by chemical carcinogens.

The effect of modifying phi chi 174 viral DNA by the chemical carcinogens beta-propiolactone, N-acetoxyacetylaminofluorene and anti-benzo[a]pyrene diol-epoxide was investigated by transfecting the modified DNA into Escherichia coli spheroplasts. Modification of the DNA in vitro by each of these agents was mutagenic for the phi chi 174 amber mutants am3 and am86. Mutagenicity depended on the induction of the "SOS" response in the host spheroplasts. Heating beta-propiolactone-treated DNA at neutral pH caused strong inactivation such that the number of lethal hits was increased 4-fold. Sucrose gradient analysis showed the induction of alkali-labile sites in the heated DNA. The "nicked circle assay" with double-stranded phi chi 174 DNA showed greater than 70% of these sites to be apurinic sites. Concomitantly with the production of these new sites, a strong increase in the mutation frequency was observed. This mutagenesis also depended upon the induction of the error-prone SOS response in the spheroplasts, as was previously shown to be the case for mutagenesis at putative apurinic sites induced directly by acid-heat treatment. These results suggest that depurination may be of importance to the mechanism of mutagenesis by beta-propiolactone and other carcinogens.

Overlapping pathways for repair of damage from ultraviolet light and chemical carcinogens in human fibroblasts.

DNA excision repair was measured in cultured human fibroblasts after single or dual treatments with ultraviolet radiation, 4-nitroquinoline 1-oxide, or N-acetoxy-2-acetylaminofluorene. Three approaches were used to monitor repair: unscheduled DNA synthesis, measured by autoradiography; repair replication, measured by the incorporation of a density-labeled DNA precursor into repaired regions; and excision of ultraviolet endonuclease-sensitive sites. When a single repair- saturating dose of one of the three carcinogens was administered, little stimulation of unscheduled DNA synthesis or repair replication could be observed by additional treatment with one of the other carcinogens. In no instance was total additivity of repair observed. These observations were confirmed by showing that the excision of endonuclease-sensitive sites produced by ultraviolet damage (i.e., pyrimidine dimers) was inhibited by exposure to 4-nitroquinoline 1-oxide and N-acetoxy-2-acetylaminofluorene. The data indicate that the repair of lesions induced by these substances may have common rate-limiting steps, a conclusion previously indicated by the repair deficiency in xeroderma pigmentosum cells in which a single mutation eliminates the repair of damage caused by each of these agents.

Defective and enhanced postreplication repair in classical and variant xeroderma pigmentosum cells treated with N-acetoxy-2-acetylaminofluorene.

Xeroderma pigmentosum (XP) cells proficient in the excision repair of pyrimidine dimers (XP variants) were also found to be proficient in the excision repair of N-2-acetoxyacetylaminofluorene (AAAF)-induced lesions in their DNA, as assayed by the photolysis of 5-bromodeoxyuridine incorporated during repair. However, the time in which the small segments of newly synthesized DNA, made immediately after treatment of cells with AAAF, were joined together to form DNA of parental size by a process called postreplication repair was long in the XP variant and classical cells. Although increasing doses of AAAF increased the time for making daughter DNA of parental size for variant and classical XP cells, AAAF did not appear to affect this process in normal human cells. Treatment of variant and classical XP cells with a relatively small dose (2.5 micron) of AAAF or 2.5 J/sq m of UV radiation several hr before a 2- to 3-fold-larger dose decreased the time for the pulse-labeled DNA to appear as parental size.

Mutagenesis of Chinese hamster cells in vitro by combination treatments with methyl methanesulfonate and N-acetoxy-2-acetylaminofluorene.

Mutational synergism was examined in Chinese hamster V79 cells exposed to methyl methanesulfonate followed by N-acetoxy-2-acetylaminofluorene (AcAAF) at different time intervals. Treatment with 500 micron methyl methanesulfonate resulted in 95% survival of cloning ability and induced approximately 4 azaguanine-resistant mutants/10(5) survivors. Seven micron AcAAF produced 10 times as many mutants, and the survival was 7%. Lethal synergism was observed for methyl methanesulfonate treatments followed by 7 micron AcAAF, and the resulting lethality was unaffected by increasing the time interval between treatments from 1 to 48 hr. However, no significant changes in the mutant frequency from that induced by AcAAF alone were found for treatment intervals of 1 to 63 hr. This result contrasts with the 6-fold enhancement of the AcAAF-induced transformation of Syrian hamster embryo cells exposed to the same combination with a 48-hr interval between treatments, as previously reported (Chem.-Biol. Interactions, 9: 351-364, 1974). The difference in the response of these two cell types demonstrates the difficulties in attempting to extrapolate the known correlation between individual mutagen and carcinogen treatments to combination treatments, with different cell types for the two cellular responses.

DNA repair in xeroderma pigmentosum cells treated with combinations of ultraviolet radiation and N-acetoxy-2-acetylaminofluorene.

We used three techniques to examine excision repair in human cells treated with ultraviolet radiation, N-acetoxy-2-acetylaminofluorene, and a combination of the two. The three techniques gave similar results. Two types of human cells were used: (a) excision repair proficient (normal human fibroblasts and xeroderma pigmentosum variants); and (b) excision repair deficient (xeroderma pigmentosum C, D, and E). Saturation doses were determined and used for combined treatments with both agents. We observed two patterns of repair: (a) in repair-proficient cells total repair was additive; and (b) in repair-deficient cells total repair was much less than additive (usually less than that repair was much less than additive (usually less than that observed for separate treatments) and N-acetoxy-2-acetylaminofluorene inhibited excision of pyrimidine dimers. We conclude that, in the first group of cells, pathways for repair of ultraviolet radiation- and N-acetoxy-2-acetylaminofluorene-induced lesions are not identical and, in the second group of cells, there is an inhibitory effect exerted by major or minor products of each agent on the repair enzyme(s) of the other.

Characterization of a quantitative assay for the in vitro transformation of normal human diploid fibroblasts to anchorage independence by chemical carcinogens.

We have characterized an assay for the quantitative measurement of the frequency of conversion to anchorage-independent growth of N-acetoxy-2-acetylaminofluorene-treated normal human diploid fibroblasts. We investigated the effects of the following parameters on the absolute number and on the frequency of anchorage-independent colonies scored: (a) the number of cells seeded per dish; (b) the type of posttreatment medium; (c) the number of population doublings allowed posttreatment prior to seeding in suspension; and (d) the carcinogen dose. The assay was linear over the range of 1.9 X 10(3) to 3.8 X 10(4) cells seeded per 6-mm dish for both total colonies scored and the induced frequency of anchorage-independent growth. The medium used posttreatment affected both the frequency and the kinetics of appearance of the anchorage-independent phenotype. The number of population doublings and the number of days allowed posttreatment prior to assaying for anchorage-independent growth potential also influenced the frequency of recovery of this phenotype. Under standardized conditions, the assay yielded a dose-response relationship for transformation to anchorage independence over the concentration range of 0 to 10 microM N-acetoxy-2-acetylaminofluorene.

DNA-protein cross-linking by chemical carcinogens in mammalian cells.

The induction of DNA cross-linking in mammalian cells by various carcinogens was investigated by the method of alkaline elution. A dose-dependent increase in DNA cross-linking was seen following exposure of human fibroblasts to N-acetyoxy-2-acetylaminofluorene and following exposure of mouse embryo cells to 7,12-dimethylbenz[a]-anthracene. No cross-link effect was seen following treatment with N-methyl-N'-nitro-N-nitrosoguanidine, benz-[a]anthracene, benz[A]anthracene-5,6-dihydroepoxide, or metabolic inhibitors. The cross-linking appeared to be DNA-protein in nature since proteinase treatment removed the effect. DNA single-strand breaks were also induced by several of these agents in the case of N-acetoxy-2-acetylaminofluorene and N-methyl-N'-nitro-N-nitrosoguanidine, approximately 70 to 90% of these breaks were rejoined after an 18-hr incubation in fresh medium, whereas repair of the cross-links induced by N-acetoxy-2-acetylaminofluorene was slight at this time.

DNA repair in V-79 cells treated with combinations of ultraviolet radiation and N-acetoxy-2-acetylaminofluorene.

Earlier experiments on human cells showed that N-acetoxy-2-acetylaminofluorene mimics ultraviolet radiation in biological and repair characteristics and that the amount of repair from a combined treatment was additive. Chinese hamster V-79 cells are less proficient than human cells in excision repair of pyrimidine dimers resulting from irradiation. We therefore investigated the combined effects of both agents on repair in V-79 cells to see whether they follow the same pattern as in human cells. They did not. Measurements of unscheduled DNA synthesis and the photolysis of DNA repaired in the presence of bromodeoxyuridine gave information about repair due to both agents, and the use of an endonuclease in an extract of Micrococcus luteus allowed us to measure repair of only ultraviolet damage in the presence of N-acetoxy-2-acetylaminofluorene damage. Each technique indicated that the amount of repair from a combined treatment was less than additive and in some cases less than that due to either agent. We conclude that V-79 cells are different from human fibroblasts in the excision repair of both ultraviolet and N-acetoxy-2-acetylaminofluorene damage and suggest that both kinds of damages inhibit repair of damage due to the other agent.

Effects of N-acetoxy-2-acetylaminofluorene and N'-nitro-N-nitrosoguanidine on nuclear DNA synthesis in rat liver.

A system for the study of DNA synthesis in isolated nuclei is described for sham and regenerating rat liver. The system has been characterized with respect to nuclear purity, conditions for optimum incorporation of [5-methyl-3H]thymidine triphosphate, time course of incorporation, product analysis by neutral and alkaline sucrose gradients, and the effect of exogenously added DNA. No difference in the basal level of activity was detected between nuclei prepared from normal or regenerating liver when isolated 24 hr after operation. However, exogenous activated DNA preferentially stimulated [5-methyl-3H]thymidine triphosphate incorporation in nuclei from regenerating liver. Activated DNA caused to react with the carcinogen N-acetoxy-2-acetylaminofluorene was a less effective primer-template in this system and decreased in a dose-dependent fashion the incorporation of [5-methyl-3H]thymidine triphosphate to below basal levels in nuclei from both normal and regenerating liver. The carcinogen N-methyl-N'-nitro-N-nitrosoguanidine had no inhibitory effect when assayed in this fashion.

DNA excision-repair deficiency of human peripheral blood lymphocytes treated with chemical carcinogens.

Human peripheral blood lymphocytes stimulated with concanavalin A for 72 hr have a 10-fold greater capacity to repair DNA damage induced by N-acetoxy-2-acetylaminofluorene than do unstimulated cells. The increased capacity of concanavalin A-activated cells to repair DNA is not observed after 24 hr in culture, a time at which stimulated cells have not begun to synthesize DNA. The maximum rate of repair synthesis obtained after treatment of stimulated cells with the "large patch"-inducing agent, N-acetoxy-2-acetylaminofluorene, is twice that obtained with methyl methanesulfonate, an agent inducing "small patch" repair. The difference between the maximum rates obtained with N-acetoxy-2-acetylaminofluorene and methyl methanesulfonate is 6-fold in a human lymphoblastoid line. Unstimulated lymphocytes show almost identical rates of repair after treatment with either N-acetoxy-2-acetylaminofluorene or methyl methanesulfonate. There is close correlation between the rate of N-acetoxy-2-acetylaminofluorene-induced repair synthesis and the loss of acetylaminofluorene adducts from DNA. Treatment of lymphocytes with methyl methanesulfonate leads to degradation of cellular DNA with the production of single-stranded regions. Such degradation is not observed with N-acetoxy-2-acetylaminofluroene. We conclude that the rate of excision repair is a function of the capacity of cells for DNA synthesis and that lymphocytes that do not synthesize DNA have a limited repair capacity and cannot be used to distinguish between large and small patch repair.

Nonspecific inhibition of DNA repair synthesis by tumor promoters in human diploid fibroblasts damaged with N-acetoxy-2-acetylaminofluorene.

The effects of selected tumor-promoting agents and their nonpromoting analogs on DNA repair synthesis were examined in human diploid fibroblasts (WI-38) damaged with N-acetoxy-2-acetylaminofluorene. Over a range of doses, three promoters (croton oil, 12-O-tetradecanoylphorbol-13-acetate, and anthralin) were found to inhibit DNA repair synthesis while their nonpromoting analogs (phorbol and 1,8-dihydroxyanthraquinone) had little effect. Another tumor promoter, phenol, inhibited DNA repair synthesis only at very high concentrations while an analog, 4-nitrophenol, produced inhibition of DNA repair synthesis at molar concentrations at which phenol had no effect. To investigate the specificity of this phenomenon, the effects of these agents on DNA-replicative synthesis, RNA synthesis, protein synthesis, and cell morphology were evaluated. At equimolar concentrations, tumor promoters were found to inhibit DNA-replicative synthesis as effectively as repair synthesis. RNA and protein synthesis were similarly inhibited over the same range of concentrations. Extensive morphological changes, interpreted as evidence of toxicity, were seen at concentrations of promoters that inhibited the macromolecular syntheses studied. The nonpromoting analogs, with the exception of nitrophenol, had little effect on these processes and showed only slight morphological damage. Thus tumor-promoting agents appeared to inhibit a number of macromolecular synthetic events, including DNA repair synthesis. It is suggested that the effect of tumor promoters on DNA repair synthesis is part of a general response to cellular injury rather than a selective response involving a single metabolic pathway. Furthermore, it is unlikely that the inhibition of repair synthesis represents the major mode of action of promoting agents in the carcinogenic process.