Cyclobutane-type pyrimidine dimers in polynucleotides.

The formation of cyclobutane-type dimers between adjacent pyrimidine residues in model polynucleotides or DNA may be represented by the general scheme See pdf 379.pdf Whereas the formation of all other known photoproducts follows the irreversible path See pdf 379.pdf Thus dimers are distinguished from other photoproducts by the fact that they can be monomerized, as well as formed, by ultraviolet irradiation. At large incident fluxes of photons the steady-state value of dimers depends on wavelength and pH, as well as on other characteristics of the surrounding medium. The number of dimers in an irradiated polynucleotide may be decreased by purely photochemical means, whereas this is not true for most other photoproducts, for which continued irradiation, irrespective of wavelength, always results in the formation of more photoproduct (37). The wavelength dependence of the steady-state for dimers is also reflected in the biological activity of irradiated transforming DNA. This experiment and the fact that photoreactivating enzyme plus visible light monomerizes dimers (and has not been demonstrated to have any effect on other photoproducts) are the strongest lines of experimental evidence that pyrimidine dimers of the cyclobutane type are biologically important lesions and can account for a large fraction of the effects of ultraviolet light on DNA in solution. Insofar as DNA is one of the more important biological structures, such dimers, when formed, account for a large part of the effects of ultraviolet radiation on biological systems.

Photoreactivation in vivo of pyrimidine dimers in paramecium DNA.

Cells of Paramecium aurelia labeled with tritiated thymidine were irradiated with ultraviolet light and then were either exposed to photoreactivating light or kept in the dark as controls. In the controls, the level of thymine-containing pyrimidine dimers did not change, but in cells exposed to photoreactivating light such dimers were destroyed. This is the first demonstration in a eukaryote of in vivo photoreactivation of thymine-containing pyrimidine dimers.

Xeroderma pigmentosum: a rapid sensitive method for prenatal diagnosis.

When normal human cells, capable of repairing ultraviolet-induced lesions in their DNA, are incubated in the thymidine analog 5-bromodeoxyuridine after ultraviolet irradiation, the analog is incorporated into the repaired regions. When such repaired cells are subsequently irradiated with 313-nanometer radiation and placed in alkali, breaks appear in the DNA at sites of incorporation of 5bromodeoxyuridine, inducing a dramatic downward shift in the sedimentation constant of the DNA. Cells from patients with the disease xeroderma pigmentosum, which causes sensitivity to ultraviolet, are incapable or only minimally capable of repair; such cells incorporate little 5-bromodeoxyuridine into their DNA under these conditions and, upon 313-nanometer irradiation and sedimentation in alkali, exhibit only minor shifts in DNA sedimentation constants. When fibroblasts developed from biopsies of normal skin and of skin from patients with xeroderma pigmentosum, as well as cells cultured from midtrimester amniotic fluid, were assayed in this fashion unequivocal differences between normal and xeroderma pigmentosum cells were shown. Xeroderma pigmentosum heterozygotes are clearly distinguishable from homozygous mutants, and results are available 12 hours after irradiation.

Permeabilization of ultraviolet-irradiated Chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus.

Chinese hamster V-79 cells were made permeable by treatment with polyethylene glycol and then incubated with a Micrococcus luteus extract containing ultraviolet-specific endonuclease activity. This treatment introduced nicks in irradiated, but not in unirradiated, deoxyribonucleic acid. The nicks remained open for at least 3 h; there was no loss of endonuclease-sensitive sites, and no excision of dimers as measured by chromatography was detected. In addition, there was no increase in ultraviolet resistance in treated cells. This suggests that the absence of a significant amount of excision repair in rodent cells is due to the lack of both incision and excision capacity.

Ultraviolet radiation-induced DNA damage and its photorepair in the skin of the platyfish Xiphophorus.

Fluence response relationships for the induction of DNA damage in the skin of UV-irradiated Xiphophorus fish were obtained by quantitative gel electrophoresis of unlabeled DNA following extraction and treatment with an enzyme preparation that makes single strand breaks next to cyclobutane pyrimidine dimers. A buffer containing 7 M urea minimized the degradation of DNA during extraction and gave reproducible results. The shapes of fluence response curves for the production of dimers by sun lamp irradiation (lambda > 290 nm) or 302 nm in the dermis of grown fish were similar. Photoreversal of dimers was readily observed by black light exposure or from the longer wavelengths (> 304 nm) from sun lamps. As expected, the number of pyrimidine dimers/incident fluence in young fish skin was considerably higher on the irradiated side of immobilized fish than it was in swimming (randomly moving) fish, and the shape of the fluence response curves was linear for all wavelengths used lambda > 290, 302, and 313 nm. On the other hand, young fish irradiated from above with lambda > 290 nm showed a less than linear relationship between pyrimidine dimers in their skin and radiation fluence because most exposure occurred on the dorsal rim of fish skin; thus, some cells in that skin were exposed to high fluences while others were not, leading to a heterogenous population of cells. Values of dimers produced were also much less than in immobilized fish. The pigment melanin decreased the number of dimers in the epidermis of grown fish exposed to lambda > 290, 302, or 313 nm, or in the dermis of fish following 302 nm, thus conferring protection against this kind of damage. No dimers were detected in the epidermis of fish exposed to 365 nm. The dimers produced at 302 and 313 nm at tumoricidal exposures correspond to 1 dimer in 10(5) base pairs.

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.

Postreplication repair in three murine melanomas, a mammary carcinoma, and a normal mouse lung fibroblast line.

Repair of ultraviolet light-induced damage to DNA was studied in three melanoma lines, a mammary carcinoma line, EMT6, and a normal lung fibroblast line, MLF, all from the mouse. The melanomas were B16CL4, a gamma-ray-resistant clonal line derived from B16; S91H-, an auxotrophic line derived from Cloudman S91; and HP, a freshly isolated line from s.c. grown Harding-Passey melanoma. The melanomas and MFL were found to perform minimal excision repair and photoreactivation. Postreplication repair, on the other hand, was an active process in all five of the lines. All three melanomas exhibited postreplication repair rates that were about twice that of MLF. The freshly isolated HP line evolved during subcultivation, and its postreplication repair rate dropped after 3 months to a rate comparable to EMT6, which was 1.5 times that of MLF. The results suggest that postreplication repair is an important process in melanomas and may be related to radiation response.

Rate and extent of DNA repair in nondividing human diploid fibroblasts.

Rates of DNA repair in ultraviolet (254 nm)-irradiated nondividing human diploid fibroblasts were determined at doses as low as 1 J/sq m using an enzymatic assay for pyrimidine dimers. In normal cells, initial rates (dimers removed per 24 hr) increased with dose to 20 J/sq m with no further increase at 40 J/sq m. At 10 J/sq m or less, repair occurred continuously over long postultraviolet periods until all the damage that could be detected was removed (for 10 J/sq m, this required 20 days; sensitivity of the assay was about 0.1 dimer/10(8) daltons). The overall rate curves appear as the sum of two first-order reactions with different rate constants (rapid, 1.7 dimers/10(8) daltons/day; slow, 0.25 dimer/10(8) daltons/day). The slow reaction extrapolates to 30 to 40% of the original dimers. Populations irradiated a second time after greater than 90% of the original damage had been removed repaired the newly added DNA damage with similar kinetics and to the same extent. Repair kinetics in a xeroderma pigmentosum strain (XP12BE, Complementation Group A, 1 J/sq m) lacks the rapid component and approximates the slow component of normal cells. If the slow component of normal cells is due to repair of less accessible dimers, as suggested by others, then by analogy, slow excision repair in XP12BE may be due to the poor accessibility of all dimers. This suggests that the XP12BE excision repair defect is in the enzymes that render dimers in chromatin accessible to repair.

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.

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.

Migration of intraperitoneally injected thyroid cells in the Amazon molly, Poecilia formosa.

We have previously reported the development of an extensive invasive growth of the thyroid gland of the gynogenetic teleost, Poecilia formosa (the Amazon molly), following i.p. injection of UV- or gamma-irradiated thyroid cells. This result was surprising by comparison with mammalian work, in which the thyroid is rarely the site for tumor metastases, but the anatomy of the circulation of fish is different from mammals, and in fish the gills and thyroid gland would be among the first tissues in which injected cells might be arrested. Techniques using a fluorescent dye, 125I membrane label, or [3H]thymidine label were used to follow the distribution of i.p. injected cells in the Amazon molly. Fish sampled as soon as 30 min after injection had some labeled cells dispersed in the connective tissue around the ventral aorta and in the bases of the gills, and by 1 to 4 hr large numbers of cells had moved into the thyroid region. A few cells still persisted there 200 hr later. Experiments on the distribution of heat-killed cells indicated that the initial distribution of the cells was largely governed by mechanical factors. Injected cells would appear to be disseminated in fish by mechanisms similar to those in mammals.

Tissue changes resulting from the injection of gamma-irradiated cells into the gynogenetic teleost, Poecilia formosa.

In previous studies a clone of the gynogenetic fish, Poecilia formosa (the Amazon molly), was successfully used as a test animal to identify ultraviolet-induced damage. This work has been extended, and the fish system was used to detect damage caused byionizing radiation. Fish cells, exposed in vitro to 250 and 500 rads, were injected into young isogenic recipients, and 9 months later the fish were examined grossly and histologically. Two of the most conspicuous changes that resulted were the development of extensive invasive thyroid hyperplasia and hypertrophy body, with an apparent reduction in the amount of hemato-poietic tissue in the head kidney and spleen. We discuss the difference between the responses of the recipient fish to cells exposed to ionizing radiation and to cells exposed to ultraviolet light.

Radiosensitivity of skin fibroblasts from atomic bomb survivors with and without breast cancer.

Fibroblasts were established in vitro from skin biopsies obtained from 55 women and 1 man with or without breast cancer and with or without exposure to radiation from the atomic bomb explosion in Hiroshima. The radiosensitivity of these cells was evaluated by clonogenic assays after exposure to X-rays or to fission neutrons from a 252Cf source. Data were fitted to a multitarget model, S/S0 = A [1 - (1 - ekD)N], for both X-ray and neutron dose-survival curves. A single hit model, S/S0 = AekD, fits the neutron dose-survival responses as well. There were no differences in the means or variances of radiosensitivity between exposed and nonexposed groups or between patients with or without breast cancer. Hence, although the sample is not large, it provides no support for the hypothesis that atomic bomb radiation preferentially induces breast cancer in women whose cells in vitro are sensitive to cell killing by radiation.

Repair of x-ray-induced single strand breaks in toluenized Escherichia coli cells.

We have used sedimentation in alkali to estimate the repair of X-ray-induced single strand breaks in the DNA of irradiated toluenized Escherichia coli cells. Extensive repair requires no exogenous cofactors except ATP although other individual NTPs (except U) or dNTPs can substitute for ATP. There is no repair in polA or resA cells and since nicotinamide mononucleotide (NMN) inhibits repair in wild type cells we interpret the results as indicating that both ligase and polymerase I are needed for repair but that the amount of any gap filling is small and extensive repair replication is not necessary.

Excision repair in ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome, and Bloom's syndrome after treatment with ultraviolet radiation and N-acetoxy-2-acetylaminofluorene.

Excision repair of damage due to ultraviolet radiation, N-acetoxy-2-acetyl-aminofluorene and a combination of both agents was studied in normal human fibroblasts and various cells from cancer prone patients (ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome and Bloom's syndrome). Three methods giving similar results were used: unscheduled DNA synthesis by radioautography, photolysis of bromodeoxyuridine incorporated into parental DNA during repari, and loss of sites sensitive to an ultraviolet endonuclease. All cell lines were proficient in repair of ultraviolet and acetoxy acetylaminofluorene damage and at saturation doses of both agents repair was additive. We interpret these data as indicating that the rate limiting step in excision repair of ultraviolet and acetoxy acetylaminofluorene is different and that there are different enzyme(s) working on incision of both types of damages.

DNA excision repair in human cells treated with ultraviolet radiation and 7,12-dimethylbenz[a]anthracene 5,6-oxide.

Excision repair was measured in normal human and xeroderma pigmentosum group C cells treated with 7,12-dimethylbenz[a]-anthracene 5,6-oxide and with ultraviolet radiation by the techniques of unscheduled DNA synthesis, repair replication, a modification of bromodeoxyuridine photolysis employing the dye Hoechst 33258 and 365 nm radiation, and endonuclease-sensitive sites assay. Radioautography and repair replication showed that in normal cells the magnitude of repair after a saturation dose of epoxide (approx. 10 microM) to be 0.1-0.2 that after a saturating ultraviolet dose (20 J/m2 at 254), though survival data showed that both doses gave nearly similar killings. Repair was of the long-patch type and repair kinetics after the epoxide treatment were similar to ultraviolet. After a combined treatment with both agents, unscheduled synthesis in normal cells was more than additive, although, considering the experimental errors, these data and those of repair replication are consistent with additivity. The epoxide did not inhibit loss of sites sensitive to the ultraviolet endonuclease. However, after a combined treatment to xeroderma pigmentosum cells there was appreciably less unscheduled synthesis than for the sum of both treatments and the epoxide inhibited the loss of nuclease-sensitive sites. We interpret the data to indicate that there are different rate-limiting steps in the removal of the ultraviolet and the epoxide damages, and that the residual repair activity in xeroderma pigmentosum cells is accomplished by different, not just fewer, enzymes than in normal cells.

Evidence that xeroderma pigmentosum cells do not perform the first step in the repair of ultraviolet damage to their DNA. 1969.

Xeroderma pigmentosum (XP) is a recessively transmitted disorder of man characterized by increased sensitivity to ultraviolet light. Homozygous, affected individuals, upon exposure to sunlight, sustain severe damage to the skin; this damage is characteristically followed by multiple basal and squamous cell carcinomas and not uncommonly by other malignant neoplasia. A tissue culture cell line was derived from the skin of a man with XP. Our measurements of ultraviolet-induced pyrimidine dimers in cellular DNA show that normal diploid human skin fibroblasts excise up to 70 per cent of the dimers 24 hours, but that fibroblasts derived from the individual with XP excise less than 20 per cent in 48 hours. Alkaline gradient sedimentation experiments show that during the 24 hours after irradiation of normal cells a large number of single-stranded breaks appear and then disappear. Such changes are not observed in XP cells. XP cells apparently fail to start, the excision process because they lack the required function of an ultraviolet-specific endonuclease. These findings, plus earlier ones of Cleaver on the lack of repair replication in XP cells, raise the possibility that unexcised pyrimidine dimers can be implicated in the oncogenicity of ultraviolet radiation.

Characterization of postreplication repair in mutagen-sensitive strains of Drosophila melanogaster.

Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by X-rays and UV radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant shocks. Postreplication repair following UV radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA byX-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine-3H. The data have been employed to construct a model for repair of UV-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed.

The mei-9 alpha mutant of Drosophila melanogaster increases mutagen sensitivity and decreases excision repair.

The mei-9(a) mutant of Drosophila melanogaster , which reduces meiotic recombination in females (Baker and Carpenter 1972), is deficient in the excision of UV-induced pyrimidine dimers in both sexes. Assays were performed in primary cultures and established cell lines derived from embryos. An endonuclease preparation from M. luteus , which is specific for pyrimidine dimers, was employed to monitor UV-induced dimers in cellular DNA. The rate of disappearance of endonuclease-sensitive sites from DNA of control cells is 10-20 times faster than that from mei-9(a) cells. The mutant mei-218, which is also deficient in meiotic recombination, removes nuclease-sensitive sites at control rates. The mei-9(a) cells exhibit control levels of photorepair, postreplication repair and repair of single strand breaks. In mei-9 cells DNA synthesis and possibly postreplication repair are weakly sensitive to caffeine. Larvae which are hemizygous for either of the two mutants that define the mei-9 locus are hypersensitive to killing by the mutagens methyl methanesulfonate, nitrogen mustard and 2-acetylaminofluorene. Larvae hemizygous for the mei-218 mutant are insensitive to each of these reagents. These data demonstrate that the mei-9 locus is active in DNA repair of somatic cells. Thus functions involved in meiotic recombination are also active in DNA repair in this higher eukaryote. The results are consistent with the earlier suggestions (Baker and Carpenter 1972; Carpenter and Sandler 1974) that the mei-9 locus functions in the exchange events of meiosis. The mei-218 mutation behaves differently in genetic tests and our data suggest its function may be restricted to meiosis. These studies demonstrate that currently recognized modes of DNA repair can be efficiently detected in primary cell cultures derived from Drosophila embryos.

Repair of ultraviolet light damage to the DNA of cultured human epidermal keratinocytes and fibroblasts.

Pure cultures of dermal fibroblasts and epidermal keratinocytes have been obtained from a single biopsy of newborn foreskin. The cells were labeled, exposed to several doses of UV light, and allowed to repair in the dark for 16 hr. The number of pyrimidine dimers before and after repair was assessed by measuring the numbers of sites in the DNA sensitive to a specific UV endonuclease. At all doses used, the extent of repair was similar in the cultured keratinocytes and cultured fibroblasts.