Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of UV-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methyl methanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of UV-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repairproficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro.Cells cultured from persons with the disease xeroderma pigmentosum (XP) are apparently defective in an early step of the excision repair process following UV irradiation, probably the incision step (1-4). However, such cells have been divided into at least five complementation groups by cell fusion (5), and the study in extracts of XP cell lines of enzyme activities believed to be involved in UV excision repair (6-10) has not adequately explained the general incision defect. As an alternative means to study this phenomenon, we have developed a cell-free system for observing repair DNA synthesis that both demonstrates the XP defect and is permeable to exogenous repair proteins. The system should provide a means of identifying and assaying for the protein(s) defective in the XP cells. In addition, by exhibiting repair DNA synthesis in reponse to carcinogens and x-rays, it should be useful for identifying both agents that induce DNA repair and cells that respond abnormally to such induction.MATERIALS AND METHODS Growth, Treatment, and Osmotic Disruption of Cells. Fibroblast lines were obtained from the American Type Culture Collection (ATCC) and from the Naval Biomedial Research Laboratory (Oakland, CA) and grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 1% antibiotic-antimycotic solutions in a humidified 5% CO2 incubator at 37°. Transfer to 6-cm plastic petri dishes was at a density of 2.5 X 105 cells per plate. All manipulation was done in dim yellow light, avoiding exposure to white light. Mycoplasma contamination was tested according to Schneider et al. (11); no contamination was found.A 6-cm plate of fibroblasts (7-20 days old) containing roughly 106 cells was washed with phosphate-buffered saline (137 mM NaCI/2.7 mM KCI/6.46 mM Na2HPO4/1.47 mM KH2PO4), the liquid was drained off, and the cells were irradiated or treated with carcinogens. One milliliter of 0.05% trypsin was added, the plates were incubated for 5 min at 370, and the cells were then transferred to a plastic tube and chilled. The plates were wa...