Normal and DNA repair-deficient human fibroblasts have been used to study induction of plasminogen activator (PA) by DNA damage. UV light induced the synthesis ofPA in skin fibroblasts of all types ofxeroderma pigmentosum (XP) in XP heterozygotes and in human amniotic cells. Enzyme induction was, however, not observed in fibroblasts of normal adults. In classical XP, which are deficient in excision repair, PA synthesis occurred in a narrow range of low-UV fluences. In such strains, the level of enzyme produced was correlated with the extent of repair deficiency. UVfluences required for PA induction in XP variants and XP heterozygotes were at least 10 times those inducing enzyme synthesis in excision-deficient XP. Maximum enzyme induction occurred 48 hr after irradiation, and the highest levels of enzyme produced were 15-20 times those of PA baseline levels. Electrophoretic analysis showed that UV irradiation enhances the synthesis ofthe Mr 60,000 human urokinase-type PA, which is present in low amounts in untreated cells. Our results suggest that PA induction in human cells is caused by unrepaired DNA damage and represents a eukaryotic SOS-like function. In addition, PA induction may provide a sensitive assay for detection of cellular DNA repair deficiencies and identification of XP heterozygotes. DNA damage induces in bacteria the coordinate expression of a set of diverse responses collectively called SOS functions. These functions include the appearance ofrepair and mutagenic activities, inhibition of septation, induction of certain prophages, and production of large amounts of the recA protein (for review, see ref. 1). Induction of SOS functions appears to involve the protease activity of the recA protein, which cleaves cellular repressors ofSOS functions including its own repressor, the lexA gene product (2-5).Several observations suggest that SOS-like activities are also (PA). PA is a highly specific serine protease closely associated with cellular transformation, neoplasia, and tumor promotion (for reviews, see refs. 16, 17). In view of the central role attributed to proteolysis in the regulation of SOS functions in bacteria and because of the possible relation of both PA and SOS-like functions with neoplastic transformation, we further explored the induction of PA in human cells by UV irradiation.In the present work, we studied the role of DNA repair in PA induction by skin fibroblasts from xeroderma pigmentosum (XP) patients. XP belongs to a class of repair-deficient human autosomal recessive disorders that have in common predisposition to cancer and chromosome instability (18,19). XP is clinically characterized by abnormally enhanced sensitivity to sunlight and the appearance of carcinomas in exposed areas of the skin. Two classes ofXP patients can be biochemically identified: those whose cells show defective excision repair of UV light-induced damage to DNA (excision-deficient XP) and patients whose cells are impaired in postreplication repair but exhibit normal excision repair (XP variants) ...

The effect of ATP on the first step of excision repair of ultraviolet damage in DNA has been studied using toluene-treated E. coli. During postirtadiation incubation, five to six times more single-strand breaks are formed in DNA in the presence of exogenous ATP than in its absence. The ATP-dependent as well as the ATP-independent endonucleolytic activities appear to be catalyzed by the same enzyme since both activities are almost completely absent in uvrA and uvrB mutants. An ATP-dependent endonucleolytic activity has been detected in nonirradiated toluene-treated E. coli. It is concluded that ATP is required in vivo for either the incision step of repair or an enzymatic reaction preceding it.Excision repair of UV (ultraviolet) damage in DNA is generally believed to proceed by means of an enzyme complex in which recognition of damage and chain incision, excision of damaged regions, repair replication, and rejoining of gaps occur together or in rapid succession (1, 2).Recently cells made permeable to deoxynucleoside triphosphates and other low-molecular-weight substances by treatment with toluene (3) have been used to study this repair process (4,5 Cultures were grown in M-9 medium, supplemented with 0.1% glucose, casamino acids (2.5 mg/ml), and thymidine (5 Ag/ml). Amino acids and biotin, when required, were added at 50 ,ug/ml and 1 /Ag/ml, respectively. To prelabel the bacterial chromosome, [at ]thymidine (5 gCi/ml, 5Ci/mmole) was added to the medium for three generations.Exponentially growing cells at a density of 0.5 to 1 X 109 cells per ml were harvested, resuspended at 5 X 109 cells per ml in 0.05 M potassium phosphate buffer (pH 7.4) and treated with toluene as described by Moses and Richardson (3).For each strain, duration of treatment with toluene which resulted in maximum stimulation of ATP-dependent DNA synthesis was employed. After toluenization, 2 X 109 cells per ml in 0.05 M potassium phosphate buffer (pH 7.4) were exposed to UV light (Mineralight lamp model no. R-51, maximum emission at 254 nm) at 15 ergs mm-2 sec at room temperature.Incubation mixtures (0.3 ml) contained 70 mM potassium phosphate buffer (pH 7.4), 13 mM M ++,5 mM NMN (nicotinamide mononucleotide), 2 X 108 irradiated or nonirradiated toluenized cells and ATP as indicated in the text. After incubation for 30 min at 370 in the dark, the reaction was terminated in the cold by addition of 1 ml of 6 mM EDTA (ethylenediaminetetraacetate)-0.05 M potassium phosphate buffer (pH 7.4). Lysis of cells and sedimentation analysis in alkaline sucrose gradients were performed as described byRupp and Howard-Flanders (7) with the exception that gradients were spun at 30,000 rpm for 120 min or 8,500 rpm for 18 hr at 4°. The number average molecular weight (M5) was calculated by computer from the distribution of radioactivity in each gradient, omitting only the top and bottom fractions which were clearly separated from the main radioactive peak.Phage T4 DNA (single-strand molecular weight of 55 X 106) was used as marker. It was assumed that the...

No abstract

We have tested human fetal fibroblasts for development associated changes in DNA repair by utilizing nucleoid sedimentation as an assay for excision repair. Among skin fibroblasts the rate of excision repair was significantly higher in non-fetal cells than in fibroblasts derived from an 8 week fetus; this was evident by a delay in both the relaxation and the restoration of DNA supercoiling in nucleoids after irradiation. Skin fibroblasts derived at 12 week gestation were more repair proficient than those derived at 8 week gestation. However, they exhibited a somewhat lower rate of repair than non-fetal cells. The same fetal and non-fetal cells were also tested for induction of the protease plasminogen activator (PA) after u.v. irradiation. Enhancement of PA was higher in skin fibroblasts derived at 8 week than in those derived at 12 week gestation and was absent in non-fetal skin fibroblasts. These results are consistent with our previous findings that in human cells u.v. light-induced PA synthesis is correlated with reduced DNA repair capacity. Excision repair and PA inducibility were found to depend on tissue of origin in addition to gestational stage, as shown for skin and lung fibroblasts from the same 12 week fetus. Lung compared to skin fibroblasts exhibited lower repair rates and produced higher levels of PA after irradiation. The sedimentation velocity of nucleoids, prepared from unirradiated fibroblasts, in neutral sucrose gradients with or without ethidium bromide, indicated the presence of DNA strand breaks in fetal cells. It is proposed that reduced DNA repair in fetal cells may result from alterations in DNA supercoiling, and that persistent DNA strand breaks enhance transcription of PA gene(s).

No abstract