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. 2022 Mar 2;119(10):e2113233119. doi: 10.1073/pnas.2113233119

Fig. 5.

Fig. 5.

Recruitment-competent p53 mutants bias DSB repair toward NHEJ over MMEJ and facilitate TT-dimer resolution. (A) Electrophoresis of PCR products from an end-joining assay using U2OS cells and Saos2 cell lines in the presence and absence of WT p53. The lower two bands were PCR products of plasmids repaired via the MMEJ pathway resulting in creation of a BstXI digestion site. The top most band was the PCR product of plasmids repaired via the NHEJ pathway that did not result in a BstXI digestion site. The numbers on top indicate percentages of repaired plasmids that were repaired via the MMEJ pathway (%MMEJ). (B) Graphical summary of A (n = 3 replicates, mean ± SD). (C) End-joining assay in control Saos2 cells (p53 null) and Saos2 cells stably expressing WT and mutant p53 in the absence and presence of UVA treatment. (D) Graphical summary of C (n = 3 replicates, mean ± SD). (E) Mutants that showed lower levels of %MMEJ correlated with higher recruitment magnitudes of p53 mutants at damage sites, and vice versa (mean ± SD). (F) DNA dot blot assay using genomic DNA extracted from Saos2 cells at different time points post-UVC treatment (Left). A duplicate blot stained with methylene blue served as a loading control (Right). (G) Normalized levels of TT dimers showed a monotonic decrease over time suggesting typical NER repair dynamics in control Saos2 cells. (H) The dot plot was probed for the unresolved TT dimers at 6 h post-UVC treatment in Saos2 cells (p53-null) and Saos2 cells expressing WT and selected p53 mutants (Left). A duplicate blot stained with methylene blue served as a loading control (Right). (I) The normalized level of unresolved TT dimers inversely correlated with the accumulation magnitude of p53 mutants. E and I share the same symbol legend. (J) Tumor sizes of Saos2 cells with and without doxycycline-induced expression of p53 WT or selected p53 mutants in immunocompromised NSG mice. Each dot represents an independent technical repeat (n ≥ 2 mice, mean± SD, *P < 0.05; **P < 0.01; ***P < 0.005). (K) Fold-change in tumor volume with and without doxycycline induction. Error bars represent SD calculated following error propagation principles.