Volume 20, Issue 9, Pages 1997-2009 (August 2017) PCAF/GCN5-Mediated Acetylation of RPA1 Promotes Nucleotide Excision Repair  Meimei Zhao, Rui Geng, Xiang Guo, Ruoshi Yuan, Xiao Zhou, Yanyan Zhong, Yanfei Huo, Mei Zhou, Qinjian Shen, Yinglu Li, Weiguo Zhu, Jiadong Wang  Cell Reports  Volume 20, Issue 9, Pages 1997-2009 (August 2017) DOI: 10.1016/j.celrep.2017.08.015 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 20, 1997-2009DOI: (10.1016/j.celrep.2017.08.015) Copyright © 2017 The Authors Terms and Conditions

Figure 1 RPA1 Is an Acetylated Protein, and the Acetylation Level Is Dramatically Induced by UV-Induced DNA Damage (A) Endogenous RPA1 was acetylated in vivo. HeLa cells were treated with 10 μM TSA and 1 mM nicotinamide for 6 hr, and then cell lysates were immunoprecipitated with anti-RPA1 antibody or normal immunoglobulin G (IgG) followed by immunoblotting with anti-RPA1 and anti-Ac-lysine antibodies. (B) Stably expressed RPA1 was acetylated in vivo. Stably expressed SFB-RPA1, SFB-Tip60, or SFB-RAD18 293T cells were treated with 10 μM TSA and 1 mM nicotinamide for 6 hr. The SFB-tagged proteins were pulled down using S protein-Sepharose and immunoblotted with anti-Flag and anti-Ac-lysine antibodies. (C) RPA1 acetylation was induced by UV irradiation. The 293T cells were treated with UV radiation (20 J/m2), IR (10 Gy), HU (1 mM), or CPT (1 μM) for 6 hr. The acetylation levels of RPA1 were detected by immunoprecipitating with anti-RPA1 antibody followed by immunoblotting with anti-RPA1 and anti-Ac-lysine antibodies. (D) PCAF acetylated RPA1 in vivo. SFB-RPA1 was cotransfected separately into 293T cells with expression vectors containing the indicated acetyltransferases. Thirty-six hours post-transfection, the cell lysates were pulled down using S protein-Sepharose, followed by immunoblotting with anti-Ac-lysine and anti-Flag antibodies. (E) PCAF acetylated RPA1 in vitro. Purified recombinant His-RPA1-WT was incubated with GST or GST-PCAF-HAT in HAT buffer with or without acetyl-CoA for 1 hr at 30°C and then immunoblotted with the indicated antibodies. (F) The acetylation level of RPA1 was reduced in PCAF knockout cells. In the presence or absence of UV irradiation, cells lysates of HeLa cells or PCAF knockout HeLa cells were immunoprecipitated with RPA1 antibody, followed by immunoblotting with RPA1 and Ac-lysine antibodies. See also Figure S1. Cell Reports 2017 20, 1997-2009DOI: (10.1016/j.celrep.2017.08.015) Copyright © 2017 The Authors Terms and Conditions

Figure 2 RPA1 Is Acetylated at Residue K163 by PCAF (A) The acetylation of RPA1 was abolished in the RPA1 D2 mutant. SFB-tagged full-length or serial deletion mutant RPA1 were transfected into 293T cells. Cell lysates were pulled down by S protein-Sepharose and immunoblotted with the indicated antibodies. (B) The acetylation of RPA1 was analyzed by mass spectrometry. SFB-RPA1 was purified from UV-irradiation-treated 293T stable cells prior to MS analysis. The MS/MS spectrum of peptide AYGAS(K#)TFGK (m/z 536.28) from SFB-RPA1 showed that lysine 163 was acetylated. (C) K163 was the major acetylation site of RPA1 in vivo. SFB-RPA1-WT or the indicated RPA1 mutations were transfected into 293T cells. SFB-tagged RPA1 was pulled down from cell lysates using S protein-Sepharose and immunoblotted with Flag and Ac-lysine antibodies. (D) PCAF acetylates RPA1 at K163 in vitro. GST-PCAF-HAT, His-RPA1-WT, and His-RPA1-K163R were expressed and purified from E. coli. In vitro acetylation assays were performed, and the reaction mixtures were immunoblotted with the indicated antibodies. The purified recombinant proteins were detected by Coomassie staining. (E) The specificity of the antibody against AcK163-RPA1 was verified by dot blot assays. The nitrocellulose membrane was spotted with the indicated amounts of RPA1 unacetylated or acetylated peptides and immunoblotted with the anti-AcK163-RPA1 antibody. (F) Verification of the specificity of anti-AcK163-RPA1 antibody. SFB-RPA1-WT or SFB-RPA1-K163R was transfected into 293T cells. SFB-tagged RPA1 was pulled down from cell lysates using S protein-Sepharose and immunoblotted with Flag and AcK163-RPA1 antibodies. (G) UV irradiation promoted acetylation of RPA1 at K163. Stably expressing SFB-RPA1 293T cells were treated with UV (20 J/m2), IR (10 Gy), HU (1 mM), and CPT (1 μM) for 6 hr, respectively. SFB-tagged RPA1 was pulled down using S protein-Sepharose and immunoblotted with Flag and AcK163-RPA1 antibodies. Cell Reports 2017 20, 1997-2009DOI: (10.1016/j.celrep.2017.08.015) Copyright © 2017 The Authors Terms and Conditions

Figure 3 K163 Acetylation of RPA1 Is Critical for RPA1-Mediated UV-Damage Repair (A) K163 acetylation of RPA1 had no effect on activation of the S phase checkpoint. HeLa cells stably expressing SFB-tagged RPA1-WT or RPA1-K163R or normal HeLa cells were transfected with RPA1 3′ UTR siRNA or control siRNA, respectively. The cells were treated with 1 mM HU for 3 hr and immunoblotted with the indicated antibodies. (B) HeLa cells stably expressing SFB-tagged RPA1-WT or RPA1-K163R or normal HeLa cells were transfected with RPA1 3′ UTR siRNA or control siRNA, respectively. Cell survival following the indicated treatment was measured by the colony formation assay. The data are presented as the mean ± SD of three independent experiments. (C) PCAF knockout HeLa cells and HeLa cells stably expressing SFB-tagged RPA1-WT or RPA1-K163 were transfected with RPA1 3′ UTR siRNA or control siRNA, respectively. After treatment with increasing doses of UV irradiation (0, 4, 8, 12, and 16 J/m2), cell survival was measured by the colony formation assay. The data are presented as the mean ± SD of three independent experiments. (D) PCAF knockout HeLa cells and HeLa cells stably expressing SFB-tagged RPA1-WT or RPA1-K163 were transfected with RPA1 3′ UTR siRNA or control siRNA, respectively. After treatment with 10 J/m2 UV irradiation and incubation for the indicated times, genomic DNAs were purified and subjected to ELISA to determine the remaining of 6-4PP (left). The remaining 6-4PP in the indicated cells was also determined by micropore UV irradiation and immunostaining (right). (E) After treatment with 2 J/m2 UV irradiation and incubation for the indicated time, genomic DNAs were purified and subjected to ELISA to determine the remaining of CPDs (left). The remaining CPD in the indicated cells was determined by micropore UV irradiation and immunostaining (right). See also Figure S2. Cell Reports 2017 20, 1997-2009DOI: (10.1016/j.celrep.2017.08.015) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Acetylation of RPA1 Increases Its Ability to Interact with XPA and Stabilizes XPA at UV-Induced DNA Damage Sites (A) K163 acetylation of RPA1 had no effect on ssDNA binding. 293T cells were transfected with Myc-tagged RPA1-WT or RPA1-K163R. At 36 hr post-transfection, the cells were irradiated with UV (20 J/m2) for 3 hr. Cell lysates were incubated with biotinylated ssDNA, and then the protein were pulled down using streptavidin-coated beads and subjected to western blot analysis with the AcK163-RPA1 and RPA1 antibodies. (B) Acetylation of RPA1 had no effect on RPA1 recruitment to DNA damaged sites. HeLa cells stably expressing SFB-tagged RPA1-WT or RPA1-K163R were transfected with RPA1 3′ UTR siRNA or control siRNA, respectively. At 48 hr post-transfection, the cells were irradiated with 20 J/m2 of UV light through an isopore polycarbonate membrane filter. After 3 hr of incubation, the cells were fixed and stained with anti-Flag antibody. (C) The acetylation of RPA1 increased its interaction with XPA. The 293T cells were transfected with SFB-tagged RPA1-WT, RPA1-K163R, RPA1-K163Q, or vector together with Myc-tagged XPA. Cell lysates were pulled down by S protein-Sepharose and immunoblotted with the indicated antibodies. (D) UV irradiation induced RPA1 acetylation and promoted its interaction with XPA. The 293T cells were transfected with SFB-tagged RPA1-WT, RPA1-K163R, or vector together with Myc-tagged XPA. At 36 hr post-transfection, the cells were irradiated with UV (20 J/m2) for 3 hr. Cell lysates were pulled down by S protein-Sepharose and immunoblotted with the indicated antibodies. (E) The endogenous coIP of RPA1 with NER factors in the presence or absence of UV damage. HeLa cells were treated with or without UV irradiation, and then cell lysates were immunoprecipitated with anti-RPA1 antibody followed by immunoblotting with the indicated antibodies. (F) Acetylation of RPA1 affected the accumulation of XPA at UV-induced damaged DNA sites. HeLa cells stably expressing SFB-tagged RPA1-WT or RPA1-K163R were transfected with RPA1 3′ UTR siRNA and Myc-XPA. At 48 hr post-transfection, the cells were irradiated with 20 J/m2 of UV light through an isopore polycarbonate membrane filter. Incubating for 3 hr, the cells were fixed and co-stained with anti-Myc antibody (XPA) and anti-Flag antibody (RPA1). See also Figures S3 and S4. Cell Reports 2017 20, 1997-2009DOI: (10.1016/j.celrep.2017.08.015) Copyright © 2017 The Authors Terms and Conditions

Figure 5 HDAC6 and SIRT1 Deacetylate RPA1 (A) Deacetylase inhibitors increased the acetylation level of RPA1 at K163. HeLa cells were treated with TSA (10 μM, 6 hr) and nicotinamide (1 mM, 6 hr), and cell lysates were immunoprecipitated with an anti-RPA1 antibody or normal IgG followed by immunoblotting with anti-RPA1 and anti-AcK163-RPA1 antibodies. (B) HDAC6 deacetylated RPA1 in vivo. The 293T cells were transfected with plasmids encoding SFB-RPA1 together with Myc-tagged HDACs (HDAC1–7). Cell extracts were precipitated (IP) using S protein-Sepharose beads. The precipitates were analyzed by immunoblotting using the indicated antibodies. (C) SIRT1 deacetylated RPA1 in vivo. The 293T cells were transfected with plasmids encoding SFB-RPA1 together with Myc-tagged SIRTs (SIRT1–7). Cell extracts were precipitated (IP) using S protein-Sepharose beads. The precipitates were analyzed by immunoblotting using the indicated antibodies. (D) HDAC6 deacetylated RPA1 in vitro. E. coli-purified recombinant His-RPA1-WT or His-RPA1-K163R was incubated with GST-PCAF-HAT in HAT buffer with acetyl-CoA for 1 hr at 30°C, MBP-HDAC6 was added to the reaction systems and incubated for 45 min at 30°C, and immunoblotting was performed with the indicated antibodies. (E) Specific HDAC6 and SIRT1 inhibitors increased the K163 acetylation level of RPA1. The 293T cells were transfected with SFB-RPA1 for 36 hr and treated with HDAC6 inhibitor (tubastatin-A, 20 μM, 6 hr) or SIRT1 inhibitor (EX527, 20 μM, 6 hr). Precipitation and immunoblotting were performed as previously indicated. Cell Reports 2017 20, 1997-2009DOI: (10.1016/j.celrep.2017.08.015) Copyright © 2017 The Authors Terms and Conditions

Figure 6 HDAC6 and SIRT1 Negatively Regulate the Accumulation of XPA at Damaged DNA Sites (A) Acetylation of RPA1 declined with a longer time of release post-UV treatment. The 293T cells were transfected with SFB-RPA1. At 36 hr post-transfection, the cells were irradiated with UV (20 J/m2) and released for the indicated time. Cell lysates were pulled down by S protein-Sepharose and immunoblotted with the indicated antibodies. (B) Inhibition of HDAC6 and SIRT1 promoted the interaction between XPA and RPA1. The 293T cells were co-transfected with SFB-RPA1 and Myc-XPA. At 36 hr post-transfection, the cells were treated with HDAC6 inhibitor and/or SIRT1 inhibitor, and RPA1 proteins were pulled down using S protein-Sepharose. The precipitates were immunoblotted with the indicated antibodies. (C) Inhibition of HDAC6 and SIRT1 promoted the accumulation of XPA at sites of UV-induced DNA damage. The HeLa cells were transfected with SFB-tagged XPA, and the cells were irradiated with 10 J/m2 UV through a micropore and incubated for the indicated times (1, 3, 6, 9 hr). The cells were also treated with or without HDAC6 inhibitor (tubastatin-A, 20 μM, 6 hr) and SIRT1 inhibitor (EX527, 20 μM, 6 hr). The immunostainings were performed with the indicated antibodies. (D) The relative fluorescence intensity of XPA was quantified with ImageJ. Fluorescence images of XPA (100 XPA foci) obtained using the same exposure time and fluorescence intensity of XPA were counted with ImageJ, and the data were normalized to the mock treatment control (∗∗p < 0.01). Cell Reports 2017 20, 1997-2009DOI: (10.1016/j.celrep.2017.08.015) Copyright © 2017 The Authors Terms and Conditions

Figure 7 DNA-PK Functions as the Upstream Signal to Promote PCAF Activation and RPA1 Acetylation upon UV Damage (A) PCAF co-localized with 6-4PP after UV irradiation. HeLa cells were transfected with the indicated plasmids. At 36 hr post-transfection, the cells were treated with UV (20 J/m2), IR (10 Gy), or HU (1 μM). The immunostainings were performed with the indicated antibodies. (B) UV irradiation promoted PCAF auto-acetylation. The 293T cells were transfected with SFB-PCAF and treated with UV irradiation, HU (1 mM), and CPT (1 μM) for 3 hr, respectively, and SFB-tagged PCAF was pulled down using S protein-Sepharose and immunoblotted with anti-Flag and anti-Ac-lysine antibodies. (C) Auto-acetylation of PCAF promoted the acetylation of RPA1. SFB-PCAF was purified from TSA/nicotinamide-treated or untreated 293T cells and incubated with His-RPA1 and acetyl-CoA in HAT buffer for 10 min. Protein levels and their acetylation status were evaluated by immunoblotting using the indicated antibodies. (D) UV damage promoted the interaction between RPA1 and PCAF but disrupted the interaction between RPA1 and HDAC6. The 293T cells were transiently transfected with SFB-RPA1 together with Myc-tagged HDAC6, PCAF, or XPA, and then the cells were treated with UV irradiation (20 J/m2). Cell lysates were pulled down by S protein-Sepharose and immunoblotted with anti-Flag and anti-Myc antibodies. (E) DNA-PK, but not ATM or ATR, was the upstream signal of UV-induced acetylation of RPA1. The 293T cells were transfected with SFB-RPA1 for 36 hr. The cells were pre-treated with ATRi (VE-821, 10 μM), ATMi (KU55933, 10 μM), or DNA-PKi (NU7441, 10 μM) for 8 hr and then irradiated with UV (20 J/m2). Incubating for 1 hr, the cell lysates were pulled down by S protein-Sepharose and immunoblotted with the indicated antibodies. (F) DNA-PKi abolished UV-induced acetylation of RPA1. The 293T cells were transfected with SFB-RPA1. The cells were pre-treated with or without DNA-PKi for 8 hr. Incubating for 1 hr, the cell lysates were pulled down by S protein-Sepharose and immunoblotted with the indicated antibodies. (G) DNA-PKi repressed UV-induced phosphorylation of PCAF. The 293T cells were transfected with SFB-PCAF. The cells were pre-treated with or without DNA-PKi for 8 hr. Incubating for 1 hr, the cell lysates were pulled down by S protein-Sepharose and immunoblotted with the indicated antibodies. (H) DNA-PKi inhibited UV-induced auto-acetylation of PCAF. The 293T cells were transfected with SFB-PCAF. The cells were pre-treated with or without DNA-PKi for 8 hr. Incubating for 1 hr, the cell lysates were pulled down by S protein-Sepharose and immunoblotted with the indicated antibodies. See also Figures S4 and S5. Cell Reports 2017 20, 1997-2009DOI: (10.1016/j.celrep.2017.08.015) Copyright © 2017 The Authors Terms and Conditions