Abstract.-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 in 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-strand 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.Xeroderma pigmentosum is an infrequently occurring human skin disorder which was shown more than 40 years ago1 to be genetically determined and to follow an autosomal recessive pattern of transmission. However, the enzymatic defect has not been elucidated until now. The skin of homozygous affected individuals appears normal at birth, but usually before age 3 severe changes consequent to sun exposure appear and relentlessly progress. Freckles of various sizes and degrees of brownness appear and are accompanied by increasing dryness, telangiectasia, atrophy, and numbers of keratoses.2 Histopathologically, there appears a combination of hyperkeratosis, marked atrophy of the dermis with irregular proliferation of certain layers, edema, dilatation of vessels, and a greatly increased accumulation of pigment.3 The eyes are affected and photophobia may be intense. Scarring of eyelids, ulceration of cornea, and blindness may occur. The changes in the skin characteristically eventuate in some form, sometimes in multiple forms, of malignant neoplasia of the skin, and metastatic epithelioma often causes death before 30. Basal cell and squamous cell epitheliomatia in large numbers may appear over the course of years. Various other forms of benign and malignant tumors of ectodermal and mesodermal origin also occur with a much increased frequency in these affected individuals.3 All these abnormalities appear to be the consequence of exposure to sun-1035

Within 12-24 hr after human cells were irradiated with ultraviolet light, approximately 50% of the ultraviolet-induced pyrimidine dimers were lost from the DNA. Pyrimidine dimers were found in the TCA-soluble fraction of ultraviolet-irradiated cells at 24 hr. Excess thymidine, caffeine, or hydroxyurea had no effect on the loss of pyrimidine dimers from the DNA of ultraviolet-irradiated cells.

Stratospheric ozone depletion may result in increased solar UV-B radiation to the ocean's upper layers and may cause deleterious effects on marine organisms. The primary UV-B damage induced in biological systems is to DNA. While physical measurements of solar UV-B penetration into the sea have been made, the effective depth and magnitude of actual DNA damage have not been determined. In the experiments reported here, UV-B-induced photoproducts (cyclobutane pyrimidine dimers) have been quantified in DNA molecules exposed to solar UV at the surface and at various depths in clear, tropical marine waters off Lee Stocking Island (23 degrees 45' N, 76 degrees 0.7' W), Exuma Cays, Bahamas. (14C)thymidine-labeled DNA or unlabeled bacteriophage phi X174 DNA was placed in specially designed quartz tubes at various depths for up to five days. Following exposure, DNA samples were removed to the laboratory where UV-B-induced pyrimidine dimers were quantified using a radiochromatographic assay, and bacteriophage DNA inactivation by solar UV-B was assayed by plaque formation in spheroplasts of Escherichia coli. Pyrimidine dimer induction was linear with time but the accumulation of dimers in DNA with time varied greatly with depth. Attenuation of dimer formation with depth of water was exponential. DNA at 3 m depth had only 17% of the pyrimidine dimers found at the surface. Bacteriophage phi X174 DNA, while reduced 96% in plaque-forming ability by a one day exposure to solar UV at the surface of the water, showed no effect on plaque formation after a similar exposure at 3 m. The data collected at the water's surface showed a "surface-enhanced dose" in that DNA damages at the real surface were greater than at the imaginary surface, which was obtained by extrapolating the data at depth to the surface. These results show the sensitivity of both the biochemical (dimers) and biological (phage plaques) DNA dosimeters. DNA dosimeters offer a sensitive, convenient and relatively inexpensive monitoring system, having both biochemical and biological endpoints for monitoring the biologically effective UV-B flux in the marine environment. Unlike physical dosimeters, DNA dosimeters do not have to be adjusted for biological effectiveness since they are sensitive only to DNA-mediated biologically effective UV-B radiation. Results of pyrimidine dimer induction in DNA by solar UV accurately predicted UV doses to the phage DNA.

A new technique has been developed for studying the extent of repair of UV-radiation damage to DNA in human cells. It is easy to use, has excellent sensitivity, and provides rapid quantitative estimates of repair. UV-irradiated cells whose DNA has been previously labeled with a radioisotope are grown after irradiation in nonradioactive bromodeoxyuridine, which is incorporated at the breaks induced by repair enzymes. After a period of growth in the thymidine analog the cells are exposed to a large flux of 313 nm radiation and then lysed on top of an alkaline sucrose gradient. Bromodeoxyuridine-containing sections of the DNA are thus selectively photolysed. Sedimentation in the alkaline gradient reveals the average molecular weight of disrupted segments and gives a measure of the number of breaks induced by repair enzymes over the whole period allowed for repair. The large change in average molecular weight observed upon exposure of normal repairing cells to 313 nm radiation is not observed in the repair-deficient cells from patients with xeroderma pigmentosum. The quantitative aspects of this assay for repair and its sensitivity should make it applicable to the study of repair induced by agents other than UV radiation.Xeroderma pigmentosum (XP) is an ultraviolet-sensitive, hereditary disease of the skin caused by an autosomal recessive mutation (1). The mutation results in a defect in the enzyme mechanism responsible for the repair of UV-induced lesions in DNA. Cleaver (2) first called attention to XP as possibly due to a repair-deficient mutation by his demonstration of decreased or absent unscheduled synthesis and repair replication in UV-irradiated "XP cells" (fibroblasts cultured from skin biopsy specimens of patients with XP). We demonstrated that XP cells do not excise UV-induced pyrimidine dimers (3), in contrast to normal cells, which can excise dimers (4). The excision process reflects early steps in repair; Cleaver (5) presented indirect evidence and we (3) provided direct evidence suggesting that a UV-specific endonuclease, the enzyme presumed to initiate the repair process, is nonfunctional in XP cells.The steps in repair are believed to be the following (6-8): (a) an ultraviolet-specific nuclease makes a single-strand break near a lesion (usually a pyrimidine dimer); (b) an exonuclease, perhaps associated with the DNA polymerase molecule, excises the dimer and other nucleotides as well; (c) DNA polymerase inserts new nucleotides into the gap produced by excision; and (d) DNA ligase closes the gap. Repair may be measured in many ways: for example, as loss of dimers from the DNA. This is a difficult measurement because of the small amounts of radioactive thymine associated with dimerized pyrimidines and the fact that the excision of dimers is only about 50% efficient in human cells (3,4). Repair may also be measured by centrifugation in alkali of DNA from normal and XP cells. After irradiation of normal cells a shift to lower molecular weight is observed, followed by the return to higher molecu...