Initial rates of DNA incision in UV-irradiated human cells differences between normal, xeroderma pigmentosum and tumour cells

Abstract

Following UV-irradiation and in the presence of inhibitors of DNA synthesis (hydroxyurea and 1-β- d-arabinofuranosylcytosine) human cells accumulate strand breaks in their DNA—as a result of enzymic incision without subsequent rejoining. We have developed a sensitive procedure which makes stringent use of these inhibitors so as to maximize the frequency of breaks detected after low levels of UV (0.25–10 Jm −2 and to permit analysis of the kinetics of break accumulation over short intervals after irradiation (up to 90 min). Since the rate of accumulation of breaks declines quickly with time of incubation (not simply as a consequence of substrate depletion), we have calculated initial rate constants by extrapolating to zero time for a range of UV doses (i.e. different substrate concentrations). Using these constants as indices of enzymic incision, we have compared a wide range of human cell types, and have (in some cases) been able to estimate the enzymatic parameters K M and V max for the incision step. Assessed in this way the human cells tested fall into a number of distinct categories. Fibroblasts from normal embryos and from xeroderma pigmentosum (XP) variant and Bloom's syndrome show high and uniform levels of incision readily distinguishable from XP(A), in turn distinct from XP(D). Tumour-derived cells and SV40-transformed fibroblasts also fall into a group with similar incision capacity, significantly lower than that of normal diploid cells. We discuss possible reasons for this distinction, and evaluate the use of inhibitors in repair studies. One of the key early steps in the repair of DNA in UV-irradiated cells is enzymic incision at damage sites. There is a well-defined group of human genetic diseases (xeroderma pigmentosum, XP) associated with high risk of malignancy in which, in all but one complementation group, the defect is located at or around incision (Robbins et al., 1974; Setlow, 1978). In bacteria incision appears to be a major site of regulation of excision repair (Grossman, 1974), and we believe that enzymatic analysis of incision in eukaryote cells, too, will provide information about the molecular nature and the control of this process. In UV-irradiated human cells, incision is readily detected by incubation with DNA synthesis inhibitors such as hydroxyurea (HU) and/or 1-β- d-arabinofurasylcytosine (araC); repair DNA synthesis is blocked, and the breaks introduced in the DNA by cellular endonucleases, which would otherwise be resealed after repair, accumulate within minutes of irradiation. This technique for studying the cellular response to UV has yielded valuable information about the capacity of various mammalian cell lines to repair DNA damage (Collins, 1977; Hiss and Preston, 1977; Collins and Johnson, 1979; Dunn and Regan, 1979; Erixon and Ahnström, 1979), and now we apply it in a quantitative comparison of different human cell lines. These include cells from normal individuals and from patients with diseases associated with DNA repair defects, and also tumour-derived and virally transformed cells. We find that different cell types fall into well defined classes according to initial apparent incision activity.