Figure 5.

Phosphorylation of S404 and S406 Are Both Important for Cellular Resistance to IR and BRCA1 Accumulation at DNA Damage Sites

(A) Generation of Abraxas knockdown U2OS cells complemented with expression of small hairpin (sh)RNA-resistant HA-tagged WT, S404A, or S406A mutants of Abraxas.

(B) Increased cellular sensitivity to IR of Abraxas-deficient cells expressing mutants of Abraxas. The colony-survival assay was carried out for cells treated with 4 Gy IR. The data are presented as means ± SD. The data analyses are processed by ANOVA and the statistical significance was determined by Tukey’s multiple comparisons test (^∗p < 0.02). There were three independent experiments that were performed (additional data are presented in Figure S5).

(C) Representative images of BRCA1 IRIF in Abra1 shRNA knockdown cells complemented with vector, WT, or mutants of Abraxas in response to 10 Gy IR followed by 2 hr incubation at 37°C.

(D) The percentage of cells containing more than ten BRCA1 IRIF foci was quantified. The data are presented as means ± SD. The data analyses are processed by ANOVA and the statistical significance was determined by Tukey’s multiple comparisons test (^∗p < 0.0001). At least three independent experiments were performed. More than 300 cells were counted for each experiment. Additional data for quantification at different time points post IR are presented in Figure S5.

(E) Quantification of the intensity of BRCA1 IR induced foci (IRIF). The data are presented as means ± SD (n > 50). The statistical analysis was carried out by Student’s t test (^∗p < 0.0002).

(F) BRCA1 accumulation at damaged chromatin depends on both S404 and S406 residues. The Orc2 was used as a marker for chromatin-bound fraction. The cells were treated with 10 Gy IR followed by 2 hr incubation at 37°C. The cellular fractionation was carried out and the chromatin fraction was analyzed (see also Figure S5).