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  • Title: Initial rates of DNA incision in UV-irradiated human cells: differences between normal, xeroderma pigmentosum and tumour cells.
    Author: Squires S, Johnson RT, Collins AR.
    Journal: Mutat Res; 1982 Aug; 95(2-3):389-404. PubMed ID: 7121492.
    Abstract:
    Following UV-irradiation and in the presence of inhibitors of DNA synthesis (hydroxyurea and 1-beta-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 KM and Vmax 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.
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