Figure 3.

NER Analysis of ERCC4 Mutants In Vivo

(A and B) In primary fibroblasts, unscheduled DNA synthesis (UDS) representing global NER activity was measured with 5-ethynyl-deoxyuridine grossly as previously described.¹²

(A) XP-F (XP42RO) and FA (1333) cells (arrows) were compared to mixed-in normal fibroblasts preloaded with polystyrene microbeads (no arrows), used as an internal control. UDS signal was quantified from 20–40 random XP-F or FA G1/G2 nuclei and expressed as a percentage of control wild-type cells.

(B) UDS signals in Ercc4^−/− MEFs measured as in (A) are expressed as a percentage of control wild-type MEFs. Ercc4^−/− cells were stably expressing an empty vector or one of various ERCC4 cDNAs (wild-type or encoding p.Leu230Pro or p.Arg689Ser).

(C) Repair kinetics of UV-light-induced DNA damage by FA-specific ERCC4 mutants in ERCC4- and NER-deficient human cells (XP2YO). Cells expressing wild-type XPF, p.Arg689Ser or p.Leu230Pro altered XPF, or XPF resulting from the 28 bp duplication were locally irradiated with UV light, cultured for the indicated times, fixed and stained for 6-4 PPs, and tagged with HA with the use of specific antibodies. Data represent the percentage of cells with 6-4 PP spots at various time points; means and SD of at least two independent experiments are shown. For each experiment, 100 cells were counted.