Abstract
The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53M/M mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.
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Acknowledgements
This work was supported by the National Basic Research Program of China (973 Program; 2012CB944500), the International Science and Technology Cooperation Program of China (2013DFG32910), the National Natural Science Foundation of China (31371491 and 30971677), and Zhejiang Provincial Natural Science Foundation of China (LZ13C120001).
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
(A) Western blot of zebrafish p53 and Δ113p53 proteins in an untreated control (ctr) and in embryos treated with γ-ray, UV irradiation (UV) and heat shock (HS) at 4 and 24 h post treatment (hpt), using A7-C10 zebrafish p53 monoclonal antibody. (PDF 625 kb)
Supplementary information, Figure S2
Visual-plus-quantitative assay systems for homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA) repairs. (PDF 167 kb)
Supplementary information, Figure S3
Knockdown of p53 and Δ113p53 proteins by p53-MO and Δ113p53-MO, or the overexpression of p53 and Δ113p53 by p53 and Δ113p53 mRNA injection in embryos injected with linearized plasmid DNA. (PDF 248 kb)
Supplementary information, Figure S4
The activation of p53 and induction of Δ113p53 proteins in zebrafish WT embryos injected with a linearized plasmid. (PDF 206 kb)
Supplementary information, Figure S5
Fluorescence imaging of HR, SSA and NHEJ repairs from zebrafish embryos injected with different reagents as indicated at 10 hpf. (PDF 614 kb)
Supplementary information, Figure S6
The induced p53M214K mutant protein and basal expression of Δ113p53p53M214K protein do not have a gain-of-function on DNA DSB repairs. (PDF 213 kb)
Supplementary information, Figure S7
Comet assay to assess the extent of DNA double-strand breaks (DSB). (PDF 131 kb)
Supplementary information, Figure S8
A TUNEL assay was used to examine apoptotic cells in Δ113p53-MO or Std-MO injected WT embryos or uninjected p53M214K mutant embryos, which were either treated with γ-ray irradiation or untreated, at 8, 16 and 24 hour post irradiation (hpi) as indicated. (PDF 523 kb)
Supplementary information, Figure S9
A TUNEL assay was used to examine apoptotic cells in Δ113p53-MO or Std-MO injected WT embryos or uninjected p53M214K mutant embryos, which were either treated with γ-ray irradiation or untreated, at 8, 16 and 24 hour post irradiation (hpi) as indicated. (PDF 212 kb)
Supplementary information, Figure S10
(A) Δ113p53 mRNA was injected into Δ113p53M/M mutant embryos at the one cell stage. (PDF 263 kb)
Supplementary information, Figure S11
Similar to zebrafish Δ113p53, human Δ133p53 was also induced only by γ-irradiation, but not by UV and heat shock. (PDF 252 kb)
Supplementary information, Figure S12
Western blot was performed to show the overexpression of p53 and Δ133p53 in H1299 cells. (PDF 201 kb)
Supplementary information, Figure S13
DNA DSB repair frequencies for HR, NHEJ and SSA were measured using Egfp positive cells sorted by a FACS machine at 24 hpt. (PDF 170 kb)
Supplementary information, Figure S14
The knockdown of Δ133p53 significantly decreased the efficiencies of HR, NHEJ and SSA DNA DBS repair pathways. (PDF 207 kb)
Supplementary information, Figure S15
Fluorescence images of γH2AX (green), RAD51 (red) and DAPI (blue) staining were taken individually and used to construct the merged picture shown in Figure 4B. (PDF 555 kb)
Supplementary information, Figure S16
FACS analysis of the percentage of cells at different cell cycle phases, based on propidium iodide (PI) staining of QSG-7701 cells transfected with siNS, p53i, Δ133p53i1 or Δ133p53i2 siRNA at different time points after 10 gray of γ-ray irradiation, as indicated. (PDF 140 kb)
Supplementary information, Figure S17
Large views for senescence-associated β-galactosidase (SA-β-gal) staining in Figure 5C to show that cell colony size was negatively correlated with cell senescence. (PDF 409 kb)
Supplementary information, Figure S18
Transcriptional expression of the indicated genes in human GSG7701 cells. (PDF 173 kb)
Supplementary information, Figure S19
A comparison of p53 responsive elements in human RAD51, RAD52 and LIG4 promoters with the known p53-repressive or -activating consensus sequences. (PDF 173 kb)
Supplementary information, Figure S20
ChIP of the p53 and Δ113p53 REs in rad51, rad52 and lig4 promoters in the absence and presence of HA-p53 and HA-Δ113p53. (PDF 234 kb)
Supplementary information, Table S1
PCR Primers (PDF 73 kb)
Supplementary information, Table S2
Antibody Information (PDF 48 kb)
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Gong, L., Gong, H., Pan, X. et al. p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage. Cell Res 25, 351–369 (2015). https://doi.org/10.1038/cr.2015.22
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DOI: https://doi.org/10.1038/cr.2015.22
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