FIGURE 5.
Effect of disrupting the interaction with PNKP on the ability of exogenous XRCC1 to correct the DNA damage-sensitive phenotype of xrcc1 mutant EM9 cells. A, the steady state levels of XRCC1, PNKP, Polβ, DNA ligase IIIα, and β-actin were determined by immunoblotting of whole cell extracts (45 μg) from: lane 1, wild-type parental AA8 cells; lane 2, xrcc1 mutant EM9 cells; lane 3, EM9 cells stably expressing Xpress-tagged wild-type XRCC1; lane 4, EM9 cells stably expressing Xpress-tagged A482T XRCC1. B, the indicated proteins were detected by immunobloting of nuclear extracts (8 μg) from lane 1, EM9 cells stably expressing Xpress-tagged wild-type XRCC1 (WT) and lane 2, EM9 cells stably expressing Xpress-tagged A482T XRCC1 (MutPNK). Nuclear extracts (80 μg) from lane 3, EM9 cells stably expressing Xpress-tagged wild-type XRCC1 (WT) and lane 4, EM9 cells stably expressing Xpress-tagged A482T XRCC1 (MutPNK) were incubated with XRCC1 antibody and the immunoprecipitates were probed for DNA ligase IIIα (DNA Lig III), XRCC1, Polβ, PNKP (PNK), and aprataxin by immunoblotting (upper panel). The results of three independent immunoprecipitation experiments are shown graphically with the error bars representing S.D. (lower panel). The amount of co-immunoprecipitated PNKP was determined by determining the ratios of co-immunoprecipitated PNKP to immunoprecipitated wild-type and mutant XRCC1 and expressing these ratios as percentage of the ratio obtained with wild-type XRCC1. C, effect of ethyl methanesulfonate (EMS) on the survival of AA8 cells (triangle), EM9 cells (circle), EM9 cells stably expressing Xpress-tagged wild-type XRCC1 (square) and EM9 cells stably expressing Xpress-tagged A482S XRCC1 (×). The graphs represent the compilation of 4 independent experiments with error bars representing S.D.