Characterization of PARP-1^−/− cells in DT40. (A) Immunoblot analysis of DT40 WT and PARP-1^−/− and PARP-1/KU70^−/− cells using anti-Parp-1, Ku70 and Rad51 antibodies. (B) Growth curves of WT cells and three different PARP-1^−/− clones. (C) Sensitivity of WT and PARP-1^−/− clones to indicated doses of ionizing radiation, monitored by colony formation assay in methylcellulose. Values of the Y-axis indicate the percent ratio of the number of colonies after IR exposure versus the number of unexposed colonies. The means of three independent experiments, and standard deviation are shown.

Differential interaction of KU70 with PARP-1 and POL-β (A) IR sensitivity of PARP-1^−/−, PARP-1/KU70^−/−, and other indicated cell lines. (B) Sensitivity of the same cell lines to continuous exposure of the indicated concentrations (nM) of CPT. (C) Sensitivity of the indicated cell lines to 1 h pulse exposure to 25 and 50 μg/μl MMS. (D) MMS sensitivity as in (C) of POL-β^−/− and POL-β/KU70^−/− cell lines. The means of three independent experiments and standard deviation are shown.

Spontaneous and CPT-induced SCE exchange. Cells were labeled with 1 mM BrdU during two cell cycle periods with (induced) or without (spontaneous) CPT treatment (2.5 nM) for the last 5.5 h. SCE in macrochromosomes of 50 nuclei were counted. The data are presented in histograms showing the frequency of nuclei with the indicated number of SCE. The mean value and standard deviation is shown in the upper right corner of each histogram.

Model of Parp function in SSB and DSB repair in DT40 cells. One branch of Parp function is the stimulation of SSB repair. Remaining base damage will be converted into DSB during DNA replication and repaired by HR. The other branch concerns the role of Parp-1 in DSB repair in which Parp-1 minimizes the inhibitory effects of Ku on HR, as proposed in this study. Both functions affect the balance between HR and NHEJ differentially, as shown in (A) WT, (B) PARP-1^−/− and (C) PARP-1/KU70^−/− cells.

Apparent absence of a PARP-2 gene in PARP-1^−/− cells. (A) Imaging of 1 mM H₂O₂ treated WT and PARP-1^−/− cells by immunofluorescence using the 10H monoclonal anti-PAR antibody. Images of PAR staining with 10H primary and FITC-labeled secondary images, and merged FITC and DAPI images are shown. (B) Measuring polymer formation in DT40 cells. WT or PARP-1^−/− cells were either untreated (a), exposed to 1 mM H₂O₂ (b), or 1 mM H₂O₂ and 3AB (c). Cells (10⁶) of each sample were permeabilized and exposed to [³²P]NAD. The cells were lysed in sample buffer and analyzed by SDS–PAGE and autoradiography; protein levels in each lane were compared by Coomassie staining (not shown). (C) Immunoblot analysis of human HeLa cells (a), DT40 WT cells (b), PARP-1^−/− cells that stably expressed human Parp-1 (c), PARP-1^−/− cells (d), or PARP-1^−/− cells that stably expressed human Parp-2 (lane e) using the indicated antibodies. (Note that human Rad51 runs with higher velocity than chicken Rad51on the SDS–PAGE). (D) Sensitivity of the indicated cell lines to increasing doses of IR assayed by colony formation, as described in Figure 1 and Materials and methods; shown are the means of three independent experiments and standard deviation.

SCneo gene conversion assay. (A) Indicated cell lines carrying the scneo construct integrated in the OVA locus were transfected with I-Sce1 endonuclease and plated in methylcellulose with or without G418. Each bar indicates the percent ratio of the number of colonies growing with and without 6418. (B) Scneo gene conversion assay, as in (A), in indicated cell lines with (black bars) or without (gray bars) coexpression of KU70 transgene. Transfection of the I-Sce1 endonuclease and Ku70 in this experiment was performed using nucleofection (AMAXA) resulting in higher transfection efficacies than in (A) (see Materials and methods). In both (A) and (B), mean values of three independent experiments and standard deviation are shown. (C) Confirmation of Ku70 overexpression by analyzing extracts of cell lines with the indicated genotypes after transfection of control or Ku70 expression vector by immunoblotting with the anti-Ku70 antibody, and an anti-cyclin B2 antibody as a loading control.

IR and CPT sensitivity in PARP/LIG4^−/− cells. (A) Confirmation of the Parp-1 deletion in LIG4^−/− cells by immunoblotting using anti-Parp-1 antibodies, and anti-cyclin B2 antibodies as a loading control. (B) Colony formation of indicated mutant DT40 cells after exposure to various doses of IR. (C) Colony formation assay of DT40 mutants in methylcellulose containing indicated concentrations of CPT. (D) Cell survival of NALM6 WT and LIG4^−/− cells after 48 h growth in liquid medium containing indicated concentrations of CPT in the presence or absence of 10 μM DPQ. In experiments B, C and D, the means of three independent experiments and standard deviation are shown. In (D), standard deviation was very low and error bars are placed within the icons representing the various cell lines.