Distribution of Repair-Incorporated Nucleotides and Nucleosome Rearrangement in the Chromatin of Normal and Xeroderma Pigmentosum Human Fibroblasts

The distribution of UV-induced DNA repair synthesis in chromatin was measured in normal human fibroblasts and in xeroderma pigmentosum (XP) fibroblasts which are partially deficient in excision repair. With normal cells we investigated the effects of hydroxyurea and UV dose on the initial distribution of nucleotides incorporated during repair synthesis and the subsequent changes in distribution during nucleosome rearrangement [Smerdon, M. J., & Lieberman, M. W. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4238]. These cells were pulse-labeled with [³H]dThd for 30 min immediately after irradiation and chased for varying times with unlabeled dThd. The initial distribution (0 chase time) of repair-incorporated nucleotides in chromatin indicated that most of these nucleotides are initially staphylococcal nuclease sensitive, and this distribution was unaffected by either the presence of 10 mM hydroxyurea or the amount of damage (3-40 J/m²). The rate at which these repair-incorporated nucleotides became increasingly nuclease resistant was also unaffected by hydroxyurea or UV dose. These data lead to two conclusions: (1) under our conditions hydroxyurea has no measurable effect on either the initial distribution of repair-incorporated nucleotides or the subsequent rate of redistribution of these nucleotides during nucleosome rearrangement; (2) if nucleosome rearrangement is induced by damage and/or the repair process, then this induction is a "local event". Cells from XP complementation groups C and D were irradiated with 3 J/m² UV, pulse-labeled for 60 min, and chased for varying times. In both groups, the initial distribution (0 chase time) indicated that most of the repair-incorporated nucleotides are initially staphylococcal nuclease sensitive. Electrophoretic data demonstrated a concurrent underrepresentation of repair synthesis in core DNA. With increasing chase time, the distribution of repair-incorporated nucleotides became more uniform in both groups of XP cells. Thus, both the initial enhanced sensitivity of repair-incorporated nucleotides to staphylococcal nuclease and the subsequent changes in sensitivity of these nucleotides during nucleosome rearrangement reported for normal cells occur in at least two complementation groups of XP cells.