A rapid procedure for measurement of DNA repair in human fibroblasts and for complementation analysis of xeroderma pigmentosum cells

Abstract

A rapid procedure for measuring unscheduled DNA synthesis has been studied in detail. Human fibroblasts were brought into the non-dividing state by either growing to confluence or starvation for arginine. Residual semi-conservative synthesis was abolished by hydroxyurea. Hydroxyurea-resistant DNA synthesis which was induced by irradiation and chemical mutagens was presumed to represent repair synthesis and provided a very rapid semi-quantitative procedure for its measurement. Problems were encountered, however, when comparing the quantitative response of different cell strains. The variability between experiments was quite large, and we found that the level of repair synthesis depended not only on the mutagen and the genotype of the cell, but also on physiological factors. This led to some anomalous results. The system was able to detect with ease the large defects in UV-induced repair synthesis in fibroblasts from patients with xeroderma pigmentosum (XP) but it would probably not easily detect less than a 50% reduction in the level of repair synthesis. By extension of this procedure, in combination with cell fusion induced by polyethylene glycol, we have developed a method for carrying out genetic complementation of XP fibroblasts, which does not entail the use of either Sendai virus or of autoradiography. Results of complementation analysis of 4 XP cell strains are presented.