95 Biochimica et Biophysica Acta, 435 (1976) 95--103 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 98596 DNA-PROTEIN CROSS-LINKING BY ULTRAVIOLET RADIATION IN NORMAL HUMAN AND XERODERMA PIGMENTOSUM FIBROBLASTS ALBERT J. FORNACE Jr. * and KURT W. KOHN Laboratory of Molecular Pharmacology, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.) (Received November 18th, 1975) Summary DNA-protein cross-linking by ultraviolet radiation was measured in human fibroblasts by an adaptation of the method of DNA alkaline elution. To measure cross-linking, a controlled frequency of DNA single-strand breaks was intro- duced by exposing the cells to a low dose of X-ray at 0°C prior to analysis by alkaline elution. The effect of prior exposure of the cells to ultraviolet radia- tion was to reduce the rate and/or extent of DNA elution from X-irradiated cells. This effect was attributed to DNA-protein cross-linking, since the effect was reversed by treatment of the cell lysates with proteinase-K. Cross-linking in normal human fibroblasts occurred immediately after ultraviolet irradiation, prior to the appearance of DNA single-strand breaks due to excision repair. Upon incubation of normal cells after exposure, to ultraviolet radiation, the cross-linking was partially repaired. In xeroderma pigmentosum cells, cross-links appeared as in normal cells, but there was no repair. Instead, the extent of cross-linking appeared to increase upon incubation after ultraviolet irradiation. Introduction Although the most extensively studied photochemical effect of ultraviolet radiation on cellular DNA has been the formation of pyrmidine dimers, this reaction may not fully account for the deleterious effect of ultraviolet radia- tion on cells [1,2]. It has been proposed that the lethal effect of ultraviolet radiation may depend also on the formation of cross-links between DNA and protein [1,3--9]. The formation of DNA-protein cross-links has been inferred from a reduction in extractability of DNA from cell lysates, and from the * Present address: Department of Pathology, 721 Huntington Avenue, Boston, Mass. 02115, U.S.A.