Therefore, although PIAS3 is definitely a regulator of the ATR pathway, it is not the key SUMO ligase for ATRIP. required for keeping the basal kinase activity of ATR prior to DNA damage. In the absence of PIAS3, ATR fails to display normal kinase activity after DNA damage, which accompanies with reduced phosphorylation of ATR substrates. Collectively, these results suggest that PIAS3 primes ATR for checkpoint activation by sustaining its basal kinase activity, revealing a new function of the PIAS family in DNA damage signaling. = 3). Immunofluorescence and Laser Micro-irradiation HeLa and U2OS cells transfected with control or PIAS3 siRNA were seeded on coverslips in 6-well plates. To detect RPA, H2AX, and ATRIP at DNA damage sites, cells were treated with CPT or irradiate with UV through 5-m filter (Millipore). Subsequently cells were pre-extracted with 0.5% Triton X-100 in PBS buffer for 3 min on ice, fixed with 3% paraformaldehyde/2% sucrose for 15 min, and then extracted again with 0.5% Triton X-100 in PBS buffer for 3 min on ice. Cells were incubated with main antibodies to RPA, H2AX, ATRIP diluted in 1 PBS comprising 3% BSA/0.05% Tween 20 for >2 h at room temperature. After 3 washes with 1 PBS comprising 0.05% Tween 20, cells were incubated with Cy3-conjugated anti-rabbit antibody and Alexa Fluor 488-conjugated anti-mouse antibody at room temperature for 1 h. Cells were then washed three times with 1 PBS comprising 0.05% Tween 20, and DNA was stained by DAPI (4,6-diamidino-2-phenylindole). To test if PIAS3 localizes to laser-induced DNA damage stripes, Biotin-HPDP U2OS cells were micro-irradiated with UV laser as previously explained (20). The pre-extraction step of immunostaining was skipped to avoid potential loss of PIAS3 from DNA damage stripes. In Vitro Kinase Assay HEK293T cells were treated with control, PIAS3, or PIAS1 siRNA for 24 h followed by transfection of Flag-ATR plasmids. Two days after plasmid transfection, Flag-ATR and Flag-ATRC were immunoprecipitated and tested with kinase assay as previously explained (21). Analysis of the UV-induced Replication Inhibition HeLa and U2OS cells transfected with control or PIAS3 siRNA were either irradiated with UV (10 J/m2) or remaining untreated. At 1 Mouse Monoclonal to Cytokeratin 18 h after UV or mock treatment, cells were labeled with 10 m EdU (5-ethynyl-2-deoxyuridine) for 30 min, trypsinized, washed with 1 PBS, and fixed in 75% ethanol at ?20 C. EdU-labeled cells were processed using Biotin-HPDP a Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay kit Biotin-HPDP according to the manufacturer’s instructions (Invitrogen). Data acquisition was performed on a FACS LSRII apparatus and analyzed with Kaluza software (Beckman Coulter). RT-Quantitative PCR (RT-qPCR) of PIAS2 mRNA Total RNA of HeLa cells transfected with control siRNA or siRNA focusing on each of the PIAS family member was isolated using PureLink RNA mini kit (Invitrogen). cDNA was synthesized using the (dT)16 primer and TaqMan Reverse Transcriptase Reagents (Existence Systems). Two primer pairs that specifically amplify (#1 ahead primer 5-GCTATTTCCTTTGCCTGGCTAT-3; #1 opposite primer 5-TTCTTCCCAATTTCTGATGCC-3; #2 ahead primer 5-CCAAGTTCAGTTGAGACTTTGC-3; #2 reverse primer 5-GTGGTGCATAGCCAGGCAA-3) were used in qPCR, which was performed using PowerUp SYBR Green Expert Biotin-HPDP Blend (Applied Biosystems) according to the manufacturer’s protocol. Reactions were analyzed by LightCycler 480 (Roche) and the relative mRNA levels were normalized to GAPDH (ahead primer 5-CGGATTTGGTCGTATTGGGC-3 and reverse primer 5-TGGAAGATGGTGATGGGATTTC-3). Results PIAS3 Is the Only PIAS SUMO Ligase Indispensable for ATR Activation While users of the PIAS family of SUMO ligases have been implicated in the DDR, whether and how they contribute to DNA damage signaling is still unclear. To address this question, we used siRNAs to knock down all 4 users of the PIAS family in HeLa cells and analyzed the effects within the CPT-induced, ATR-mediated Chk1 phosphorylation at Ser317. The knockdown of PIAS1, PIAS3, and PIAS4 was confirmed by Western blot (Fig. 1mRNA levels were determined by RT-qPCR. and and and and < 0.05. < 0.05. and < 0.05. PIAS3 Is definitely Dispensable for ATRIP SUMOylation Our earlier studies showed that ATRIP is definitely progressively SUMOylated after UV treatment, and that ATRIP SUMOylation is required for efficient activation of the ATR pathway (20). To test if PIAS3 or another PIAS.
Therefore, although PIAS3 is definitely a regulator of the ATR pathway, it is not the key SUMO ligase for ATRIP
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