PARP1 and CHK1 coordinate PLK1 enzymatic activity during the DNA damage response to promote homologous recombination-mediated repair

Abstract Polo-like kinase 1 (PLK1) is a master kinase that regulates cell cycle progression. How its enzymatic activity is regulated in response to DNA damage is not fully understood. We show that PLK1 is enriched at double strand breaks (DSBs) within seconds of UV laser irradiation in a PARP-1-dependent manner and then disperses within 10 min in a PARG-dependent manner. Poly(ADP-)ribose (PAR) chains directly bind to PLK1 in vitro and inhibit its enzymatic activity. CHK1-mediated PLK1 phosphorylation at S137 prevents its binding to PAR and recruitment to DSBs but ensures PLK1 phosphorylation at T210 and its enzymatic activity toward RAD51 at S14. This subsequent phosphorylation event at S14 primes RAD51 for CHK1-mediated phosphorylation at T309, which is essential for full RAD51 activation. This CHK1–PLK1–RAD51 axis ultimately promotes homologous recombination (HR)-mediated repair and ensures chromosome stability and cellular radiosensitivity. These findings provide biological insight for combined cancer therapy using inhibitors of PARG and CHK1.


Supplementary figures and legends
. Recruitment of polo-like kinases to the UV laser-induced stripes (A) Schematic representation of Fucci cell-cycle labeling. A fluorescent probe that labels individual G1 phase nuclei in red (mKO2-hCdt1) and S/G2/M phase nuclei green (mAG-hGeminin).
(B) PLK1 recruitment was correlative with its expression. Fucci HeLa cells expressing Cdt1-mKO2 only or expressing Geminin-mAG1 only were subjected to UV laser-microirradiation treatment before PLK1 and γ-H2AX analysis by immunofluorescence.
(D) Epitope-tagged PLK1 enrichment at ER-AsiS1-restriction-enzyme-induced DSB. ER-AsiSI U2OS cells transiently expressing FLAG-VEC or FLAG-PLK1 for 48 h were treated or not with 4-OHT (300 nM) for 2h or 4 h, and then analyzed by ChIP using a FLAG antibody. The data represent the means ± SD. **p <0.01.   (D) The dependency of phosphorylation on the CHK1-PLK1 interaction. GST-PLK1 PBD was used to pulldown endogenous CHK1 from total 293T cell lysates in the presence or absence of CIP, or heatinactivated CIP (100℃ for 10 min). Asterisk represent the degradation band of GST-PLK1 PBD.
(E)The suitability of a phospho-specific pS137 PLK1antibody for immunoprecipitating endogenous PLK1 phosphorylated at S137. Total cell lysates derived from 293T cells pretreated or not with nocodazole (340 nM) for 16 h were immunoprecipitated with the pS137 PLK1antibody and analyzed by immunoblotting with a PLK1 antibody.
(F) The suitability of a phospho-specific pS137 PLK1 antibody for immunoprecipitating FLAG-PLK1 phosphorylated at S137. 293T cells transiently expressing FLAG-VEC or FLAG-PLK1 pretreated or not with nocodazole (340 nM) for 16 h, were immunoprecipitated with the pS137 PLK1antibody and analyzed by immunoblotting.
(G) The effect of UCN-01 on the X-ray radiation-induced surge of PLK1 phosphorylation at S137. Total cell lysates derived from 293T cells pretreated with UCN-01 (10 nM) for 60 min before X-ray radiation (10 Gy), then immunoprecipitated after 30 min with a pS137 PLK1 antibody and analyzed by immunoblotting.
(H) DNA damage-induced PLK1 phosphorylation at S137 after Olaparib treatment. 293T cells were pretreated with Olaparib (1 µM) for 60 min before X-ray radiation (10 Gy), total cell lysates derived from these cells 30 min after IR were immunoprecipitated after 30 min with pS137 PLK1 antibody followed by immunoblotting with antibodies as indicated.   (G) The role of CHK1 and PLK1 on HR. DR-U2OS cells stably expressing FLAG-PLK1 S137D were infected with I-SceI lentivirus for 36 h. Cell were pre-treated with UCN-01 (10 nM), BI2536 (10 nM), gallotannin (10 µM), UCN-01 plus BI2536, or UCN-01 plus gallotannin for 12 h before harvest. The percentage of GFP positive cells and western blotting were shown in G.
(J and K) Representative images (K) and immunoblot analysis (J) of chromosome aberrations upon RAD51 depletion or re-expression with its variants (shown in Figure 7H). RAD51 was depleted by transfecting HeLa cells stably expressing the indicated constructs with siRAD51 (3'UTR). The transfectants were X-ray radiated (5 Gy) and then exposed to colchicines (0.4 µg/ml) for 6 h before harvesting. The cells were analyzed by immunoblotting with the indicated antibodies (J). Chromosome spreads were performed and images were captured under a confocal imaging system. Blue arrows (others), dicentric, deletion, ring; green arrows, fusions; red arrows, breaks.
(L) Immunoblot analysis of the cell samples shown in Figure 7I. RAD51 was depleted by transfecting HeLa cells stably expressing the indicated constructs with siRAD51 (3'UTR). Total cell lysates were extracted 48 h after transfection and analyzed by immunoblotting.
All data are derived from three independent experiments.