Fig 1: Summary figure of DSB repair in G1 phase.(i) DSB occurs in a cell in G1 phase that is expressing 8E6. (ii) NHEJ initiates. DNA-PKcs is recruited to the lesion where it is auto-phosphorylated. (iii) NHEJ stalls due to 8E6 mediated p300 degradation. This leaves unresolved pDNA-PKcs repair complexes [9]. (iv) HR initiates at the site of failed NHEJ. The MRN complex, CtIP, and EXO1 resect one strand of DNA near the lesion, producing single stranded DNA [73]. RPA complexes coat and stabilize the resulting single stranded DNA [39]. RPA70 foci colocalize with pDNA-PKcs indicating that HR-mediated DNA resection occurs after NHEJ fails. Then, RAD51 is recruited to the break site. (v) HR cannot be complete due to the lack of a homologous template and/or 8E6-mediated inhibition of HR [7]. This causes deletions due to antagonist DNA end process by NHEJ and HR and/or by failure to complete HR after resection. (vi) The failure of NHEJ causes cells to use tertiary DSB repair pathways to fix the lesion. (vii) This alternative repair pathway (e.g., Alt-EJ) is error prone and increases other types of mutations (such as SNPs).
Fig 2: By binding p300, 8E6 allows RAD51 and RPA70 foci to colocalize with persistent pDNA-PKcs foci.(A) Representative images of HFK LXSN and HFK 8E6 cells stained for RPA70 (green) and pDNA-PKcs (red) following zeocin treatment (10 µg/mL, 10 min). (B) Percentage of HFK cells with colocalized RPA70 and pDNA-PKcs foci. (C) Representative images of HFK LXSN and HFK 8E6 cells stained for RAD51 (green) and pDNA-PKcs (red) following zeocin treatment (D) Percentage of HFK cells with colocalized RAD51 and pDNA-PKcs foci. (E) Percentage of primary HFKs with colocalized RPA70 and pDNA-PKcs foci. (F) Percentage of primary HFKs with colocalized RAD51 and pDNA-PKcs foci. White arrow indicates colocalization. All values are represented as mean ± standard error. The statistical significance of differences between two groups were determined using Student’s t-test. * indicates significant difference between 8E6 and LXSN with same treatment (p< 0.05). # indicates a significant difference (p < 0.05) between control (solvent) and treated group within each cell line. At least 150 cells were counted over three independent experiments. Nuclei were determined by DAPI staining. The edge of this staining is shown by a white line depicting the nucleus.
Fig 3: p300 reduces co-localization of RAD51 and RPA70 foci with pDNA-PKcs foci.(A-B) Percentage of HFK WT and p300 KO cells with (A) colocalized RPA70 (green) and pDNA-PKcs (red) foci or (B) RAD51 (green) and pDNA-PKcs (red) foci over a 32-hour time course following zeocin exposure (10 µg/mL, 10 min). (C-D) Percentage of CCS1477 (1 µM) or DMSO treated HFK LXSN cells that contained colocalized (C) RPA70 (green) and pDNA-PKcs (red) or (D) RAD51 (green) and pDNA-PKcs (red) foci following zeocin treatment. (E-F) Percentage of primary HFK cells treated with CCS1477 or DMSO that contained (E) colocalized RPA70 and pDNA-PKcs foci 16-hours following zeocin treatment or (F) RAD51 and pDNA-PKcs foci 24-hours following zeocin treatment. All values are represented as mean ± standard error. The statistical significance of differences between cell lines or treatments were determined using Student’s t-test. * indicates significant difference between two groups (A-D) or LXSN and 8E6 (E-F) with same treatment (p< 0.05). # indicates p < 0.05 between control (0 h) and zeocin treated group within each cell line. At least 150 cells were counted over three independent experiments.
Fig 4: Proper acetylation and deacetylation of human RPA promote RPA and RAD51 loading. (A) Immunoprecipitation and western blot showing acetylation of the ectopically expressed RPA1 in response to the DNA damage in HEK293T cells. An anti-acetyl-lysine antibody was used to carry out immunoprecipitations, followed by probing with an anti-FLAG antibody. (B) Immunoprecipitation showing acetylation of the ectopically expressed WT or 3KR-RPA1 in HEK293T cells. (C) Western blot indicating RPA1 levels in HEK293T cells transfected with the control shRNA or in cells with a vector or reconstituted WT, 3KQ or 3KR RPA1. GAPDH was used as a loading control. (D, E) Immunostaining and quantification of RPA1 or RAD51 foci number post VP16 treatment (5 µM, 1 h) in HeLa cells. ?H2AX was stained as a marker of the DNA damage, and the relative RPA and RAD51 foci number was normalized to the corresponding ?H2AX foci number. Scale bar: 10 µm. (F, G) An ssDNA pull-down assay and the quantification showing the effect of 3KQ or 3KR mutation on the ssDNA-binding affinity of human RPA. Error bars indicate the standard deviation from three independent experiments. Statistical analysis was calculated with the Student's t-test. n.s., no significance. * P< 0.05, ** P< 0.01.
Fig 5: Proper acetylation and deacetylation of human RPA promote accurate DSB repair by HR while suppressing the SSA or alt-EJ pathway. (A–D) Plot showing the relative efficiency of HR, BIR, alt-EJ, and SSA, respectively, measured with the corresponding GFP reporters for indicated cell lines. The corresponding RPA1 protein level was detected by Western blot and shown at the bottom of each plot. GAPDH served as a loading control. (E) Representative images showing micronuclei in indicated Hela cells. Arrows indicate micronuclei stained by DAPI. The level of micronuclei in the reconstituted WT, 3KQ or 3KR cells was quantified. Quantifications of A–E are the average values of three independent experiments. Data were analyzed by Students’ t-test. n.s., no significance. * P< 0.05, ** P< 0.01. Scale bar: 10 µm. (F) Survival curves for RPA1 siRNA cells complemented with the plasmid expressing the WT, 3KQ or 3KR plasmid upon exposure to iniparib treatment. Data were analyzed by two-way ANOVA, n = 3. *P< 0.05, ** P< 0.01.
Supplier Page from Abcam for Anti-RPA70 antibody [8C3-D12-H10]