Effects of inhibitors on repair of DNA in normal human and xeroderma pigmentosum cells after exposure to x-rays and ultraviolet irradiation

Experiments were carried out to obtain direct evidence for the hypothesis that in human cells the repair of UV-damaged DNA is initiated by an incision step, and that this step is defective in cells from patients having Xeroderma pigmentosum (XP). The alkaline sucrose gradient centrifugation technique was used to detect breaks in the DNA.A decreased sedimentation velocity of the DNA was found after exposure of normal and XP cells to high doses of UV (5000 erg/mm²). Breaks were induced in the DNA by the UV irradiation without the action of an enzyme. After exposure of both types of cell to UV doses of 100–500 erg/mm², breaks that might occur by enzymic incision were not observed, possibly because of immediate rejoining.After single-strand breaks had been induced by X-rays, rejoining did not occur at temperatures lower than 22°. Rejoining was inhibited by KCN, 2,4-dinitrophenol, EDTA, iodoacetate and crystal violet. Actinomycin D, acriflavine and phleomycin, also tested as potential inhibitors of the repair process, induced breaks or conformational changes in the DNA of unirradiated normal and XP cells.Application to UV-exposed cells of conditions that inhibit the rejoining of breaks did not cause accumulation of breaks in the DNA. The results suggest a coordinated and sequential performance of the steps in the repair of each UV lesion by repair enzymes which may act as a complex.