UV-triggered p21 degradation facilitates damaged-DNA replication and preserves genomic stability

Although many genotoxic treatments upregulate the cyclin kinase inhibitor p21, agents such as UV irradiation trigger p21 degradation. This suggests that p21 blocks a process relevant for the cellular response to UV. Here, we show that forced p21 stabilization after UV strongly impairs damaged-DNA replication, which is associated with permanent deficiencies in the recruitment of DNA polymerases from the Y family involved in translesion DNA synthesis), with the accumulation of DNA damage markers and increased genomic instability. Remarkably, such noxious effects disappear when disrupting the proliferating cell nuclear antigen (PCNA) interacting motif of stable p21, thus suggesting that the release of PCNA from p21 interaction is sufficient to allow the recruitment to PCNA of partners (such as Y polymerases) relevant for the UV response. Expression of degradable p21 only transiently delays early replication events and Y polymerase recruitment after UV irradiation. These temporary defects disappear in a manner that correlates with p21 degradation with no detectable consequences on later replication events or genomic stability. Together, our findings suggest that the biological role of UV-triggered p21 degradation is to prevent replication defects by facilitating the tolerance of UV-induced DNA lesions.

served to visualize the whole cell and nuclei respectively. 200 GFP positive binucleted cells were scored. The percentage of cell that had entered mitosis was calculated as the number of binucleated cells (arrested prior to cytokinesis by cytochalasin B) with respect to the total cells including both binucleated and mononucleated cells (cells that have not entered mitosis during the cytochalasin B treatment).
Cell Viability and clonogenic assay: To evaluate cell survival, U2OS cells were transfected, replated at low density and UV irradiated 1 day later. 72hs after irradiation transfected f-GFP positve cells were scored as viable or non-viable according to its nuclear structure and shape. 300 cells were counted in three independent experiments. For the clonogenic assay U2OS cells were transfected in 12-well plates, trypsinized 24hours later and replated onto 60mm dishes in duplicates at a density of 250 cells per plate. The following day cells were UV irradiated at the indicated doses and incubated for 8-10 days. Colonies formed were fixed and stained with crystal violet and then counted in duplicates (colonies with more than 50 cells were scored as positive).
γH2AX quantification: To measure the average intensity of γH2AX in a given sample, photographs of random fields for γH2AX, p21 and DAPI were taken with a Zeiss Axioplan confocal microscope with the 60X water objective. All images within each experiment were acquired with the same exposure time and instrument settings and were processed equally without any further modification. Using the Image J software, nuclei were identified and delimited using the DAPI signal and transfected cells were selected using the p21 staining.
The intensity of γH2AX in all transfected nuclei (either pan nuclear or focal) was calculated using the measure tool of the image J software.

Supplementary Figures
Supplementary Figure 1: Persistent p21/PCNA interaction after UV irradiation causes increased cell death after UV irradiation. A) HCT116 p21 +/+ and p21-/-cells were UV irradiated (10 J/m 2 ) and 53BP1 foci accumulation was determined 4 hours later as described in Figure 2E. Representative panels of HCT 116 cells with and without 53BP1 focal organization are shown on the left. B) HCT116 p21 +/+ and p21-/-cells were UV irradiated (10 J/m 2 ) and the micronuclei assay was performed as described in Figure  with CldU and IdU following the protocol described in Figure 6A. Representative fields of labeled DNA fibers from HCT116 p21+/+ and p21-/-unirradiated and UV irradiated cells. Figure 7: Inefficient p21 degradation after UV irradiation impairs DNA replication and karyokinesis. A) U2OS cells co-transfected with the indicated p21 constructs and fGFP were or were not UV irradiated (5J/m2) and 24 hrs later EdU incorporation was performed for 15 minutes before fixation. Different fields were used to establish the number of EdU (+)/ transfected cells. B) Representative panels of UV irradiated fields are shown C) Experiments similar to the one described in A) were performed for HCT116 p21+/+ and -/cells. D). U2OS cells transfected with the indicated p21 constructs were UV irradiated and the percentage of cells that reached Karyokinesis (and therefore finished S-phase and surpassed anaphase) was calculated using the CBPI. Representative mono and binucleated cells are shown. E) The number of binucleated cells/ transfected cells was determined scoring 200 transfected cells in three independent experiments. Significance of the differences between E.V. and each condition *: p<0.05, no asterisk= NS -not significant, p>0.05.