UV-Induced RPA1 Acetylation Promotes Nucleotide Excision Repair Hanqing He, Jiajia Wang, Ting Liu  Cell Reports  Volume 20, Issue 9, Pages 2010-2025 (August 2017) DOI: 10.1016/j.celrep.2017.08.016 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 20, 2010-2025DOI: (10.1016/j.celrep.2017.08.016) Copyright © 2017 The Authors Terms and Conditions

Figure 1 RPA1 Is Acetylated by GCN5 and PCAF (A) HEK293T cells were transiently transfected with plasmids encoding SFB-tagged RPA1 together with plasmids encoding Myc- or HA-tagged acetyltransferases. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. (B) GCN5 acetylates RPA1 in a dose-dependent manner. HEK293T cells were transiently transfected with plasmids encoding SFB-tagged RPA1 together with increasing amounts of plasmid encoding Myc-tagged GCN5. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. (C) The enzymatically inactive mutant of GCN5 fails to acetylate RPA1. HEK293T cells were transiently transfected with plasmids encoding SFB-tagged RPA1 together with plasmids encoding Myc-tagged wild-type GCN5 or its mutant YA/FA. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. (D) Endogenously expressed RPA1 is acetylated in vivo. HeLa cells were either mock treated or treated with nicotinamide (10 mM) and TSA (10 μM) for 4 hr before they were collected. Cell lysates were then incubated with protein A agarose beads conjugated with RPA1 antibody, and western blot analysis was performed with indicated antibodies. (E) GCN5 acetylates RPA1 in vitro. His-tagged RPA1, GST-tagged GCN5-HAT, or GST alone were purified from E. coli. The purified proteins were incubated in reaction buffer in the presence or absence of the acetyl-CoA (2 mM) at 37°C for 1 hr. Upper panel: RPA1 acetylation was detected by immunoblotting. Lower panel: purified proteins visualized by Coomassie staining are shown. (F) HEK293T cells were transiently transfected with plasmids encoding SFB-tagged RPA1 together with plasmids encoding Myc-tagged GCN5 or PCAF. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. (G) Association of endogenous RPA1 with GCN5 and PCAF. Cell lysates were incubated with protein A agarose beads conjugated with anti-RPA1 antibody, and western blot analysis was performed according to standard procedures. (H) Direct in vitro binding between recombinant His-RPA1 and GST-GCN5 or GST-PCAF purified from E. coli. GST serves as negative control for RPA1 binding. (Upper panel) RPA1 was detected by immunoblotting. (Lower panel) Purified proteins visualized by Coomassie staining are shown. Cell Reports 2017 20, 2010-2025DOI: (10.1016/j.celrep.2017.08.016) Copyright © 2017 The Authors Terms and Conditions

Figure 2 RPA1 Is Acetylated at Lysine 163 (A) Schematic representation of RPA1 mutants used in this study. (B) HEK293T cells were transiently transfected with indicated plasmids for 24 hr. Cell lysates were immunoprecipitated with anti-Myc antibody, and western blot analysis was performed with indicated antibodies. (C) GCN5 acetylates RPA1 at K163 in vivo. HEK293T cells were transiently transfected with the indicated plasmids for 24 hr. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. (D) The K163R mutant interacts with GCN5 as efficiently as wild-type RPA1. HEK293T cells were transiently transfected with indicated plasmids for 24 hr. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. (E) RPA1 is acetylated at K163. HEK293T cells transfected with the indicated plasmids were either mock treated or treated with nicotinamide (10 mM) and TSA (10 μM) for 4 hr before they were collected. Cell lysates were then incubated with protein A agarose beads conjugated with anti-Flag antibody, and western blot analysis was performed with indicated antibodies. (F) GCN5 acetylates RPA1 at K163 in vitro. Purified wild-type RPA1 or the K163R mutant were incubated with purified GCN5-HAT in reaction buffer in the presence or absence of the acetyl-CoA (2 mM) at 37°C for 1 hr. (Upper panel) RPA1 acetylation was detected by immunoblotting. (Lower panel) Purified proteins visualized by Coomassie staining are shown. (G) Sequence alignment of the region containing the acetylation site in RPA1 from different species. Cell Reports 2017 20, 2010-2025DOI: (10.1016/j.celrep.2017.08.016) Copyright © 2017 The Authors Terms and Conditions

Figure 3 HDAC6 and SIRT1 Mediate RPA1 Deacetylation (A and B) HDAC6 (A) and SIRT1 (B) mediate RPA1 deacetylation. HEK293T cells were transiently transfected with the indicated plasmids for 24 hr. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. (C and D) The enzymatically inactive mutant of HDAC6 (2HA; C) or SIRT1 (HY; D) failed to deacetylate RPA1. HEK293T cells were transiently transfected with the indicated plasmids for 24 hr. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. (E) HDAC6 deacetylates RPA1 in vitro. Bacterially purified RPA1 was in vitro acetylated by recombinant GCN5 HAT domain. Following this, acetylated RPA1, as a substrate, was incubated with recombinant HDAC6 at 37°C for 2 hr. The reaction mixtures were then subjected to SDS-PAGE and immunoblotted with anti-acetyl lysine antibody (upper panel). Purified proteins visualized by Coomassie staining are shown (lower panel). (F) SIRT1 deacetylates RPA1 in vitro. Bacterially purified RPA1 was in vitro acetylated by recombinant GCN5 HAT domain. Following this, acetylated RPA1, as a substrate, was incubated with recombinant SIRT1 in the presence of 5 mM NAD+ at 37°C for 2 hr. The reaction mixtures were then subjected to SDS-PAGE and immunoblotted with anti-acetyl lysine antibody (upper panel). Purified proteins visualized by Coomassie staining are shown (lower panel). (G) RPA1 interacts strongly with HDAC6. HEK293T cells were transiently transfected with plasmids encoding SFB-tagged RPA1 together with plasmids encoding Myc-tagged SIRT1 or HDAC6 for 24 hr. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. (H) Direct in vitro binding between recombinant His-RPA1 and MBP-HDAC6 or MBP-SIRT1 purified from E. coli. MBP serves as negative control for RPA1 binding. (Upper panel) RPA1 was detected by immunoblotting. (Lower panel) Purified proteins visualized by Coomassie staining are shown. (I) Association of endogenous RPA1 with HDAC6. Cell lysates were incubated with protein A agarose beads conjugated with anti-RPA1 antibody, and western blot analysis was performed according to standard procedures. (J and K) Depletion of HDAC6 (J) or inhibition of HDAC6 activity (K) increases RPA1 acetylation. HeLa cells were infected with lentiviral shRNA constructs that target HDAC6 for 48 hr or were treated with HDAC6 inhibitor tubacin (20 μM) for 6 hr. Cell lysates were incubated with protein A agarose beads conjugated with anti-RPA1 antibody, and western blot analysis was performed with indicated antibodies. Cell Reports 2017 20, 2010-2025DOI: (10.1016/j.celrep.2017.08.016) Copyright © 2017 The Authors Terms and Conditions

Figure 4 UV Irradiation Stimulates RPA1 Acetylation (A) HeLa cells were treated with 1 μM CPT, 5 mM HU, or 40 J/m2 UV for 4 hr. Cell lysates were incubated with protein A agarose beads conjugated with anti-RPA1 antibody, and western blot analysis was performed with indicated antibodies. (B) HeLa cells were treated with the indicated doses of UV irradiation and harvested 4 hr later. Cell lysates were incubated with protein A agarose beads conjugated with anti-RPA1 antibody, and western blot analysis was performed with indicated antibodies. (C) HeLa cells were treated with 40 J/m2 UV and allowed to recover for the indicated times. Cell lysates were incubated with protein A agarose beads conjugated with anti-RPA1 antibody, and western blot analysis was performed with indicated antibodies. (D) UV irradiation triggers RPA1 acetylation at lysine 163. HeLa cells stably expressing HA-Flag tagged wild-type RPA1 or K163R mutant were treated with 40 J/m2 UV for 4 hr. Cell lysates were incubated with protein A agarose beads conjugated with anti-Flag antibody, and western blot analysis was performed with indicated antibodies. (E) GCN5 and PCAF are required for UV-induced RPA1 acetylation. HeLa cells infected with indicated lentiviral shRNAs were treated with 40 J/m2 UV for 4 hr. Cell lysates were incubated with protein A agarose beads conjugated with anti-RPA1 antibody, and western blot analysis was performed with indicated antibodies. (F) UV irradiation has no effect on the interaction between RPA1 and GCN5. HEK293T cells were transiently transfected with the indicated plasmids for 24 hr. Cells were then treated with 40 J/m2 UV and allowed to recover for 4 hr. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. (G) UV irradiation disrupts the interaction between RPA1 and HDAC6. HEK293T cells were transiently transfected with the indicated plasmids for 24 hr. Cells were then treated with 40 J/m2 UV and allowed to recover for 4 hr. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with indicated antibodies. (H and I) UV irradiation facilitates translocation of HDAC6 from the nucleus to the cytoplasm. HeLa cells transfected with Flag-tagged HDAC6 were either mock treated or treated with 40 J/m2 UV for 4 hr before fixing and processed for HDAC6 immunofluorescence (H). The nucleus/cytoplasm ratio of HDAC6 was quantified using NIH ImageJ software (I). (J) HeLa cells were treated with 40 J/m2 UV for 4 hr before they were collected. Cell lysates were separated into cytoplasmic and nuclear fractions. HDAC6 levels were analyzed by an anti-HDAC6 western blot. Anti-GAPDH and anti-PARP1/anti-MCM3 western blot analyses were also performed to detect the purity of cytoplasmic and nuclear fractions, respectively. Cell Reports 2017 20, 2010-2025DOI: (10.1016/j.celrep.2017.08.016) Copyright © 2017 The Authors Terms and Conditions

Figure 5 RPA1 Acetylation Is Required for Optimal Repair of UV-Induced DNA Damage (A) HeLa cells stably expressing HA-Flag-tagged wild-type RPA1 or the K163R mutant were infected with lentiviral shRNA constructs that target the 3′ UTR of RPA1 and cultured in medium containing puromycin for 2 days. The levels of the indicated proteins were analyzed by western blot. (B) RPA1 acetylation has no effect on assembly of the RPA complex. HeLa cells stably expressing HA-Flag-tagged wild-type RPA1 or the K163R mutant were infected with lentiviral shRNA constructs that target the 3′ UTR of RPA1 and cultured in medium containing puromycin for 2 days. Cells were then either mock treated or treated with 40 J/m2 UV and allowed to recover for 4 hr. Cell lysates were incubated with protein A agarose beads conjugated with anti-Flag antibody, and western blot analysis was performed with indicated antibodies. (C) RPA1 acetylation is not required for its recruitment to sites of DNA damage. HeLa cells stably expressing HA-Flag-tagged wild-type RPA1 or the K163R mutant were infected with lentiviral shRNA constructs that target the 3′ UTR of RPA1 and cultured in medium containing puromycin for 2 days. Cells were then either mock treated or treated with 40 J/m2 UV, 1 μM CPT, or 5 mM HU and allowed to recover for 1 hr. Cells were then fixed and processed for RPA1 immunofluorescence. (D) Mutation of the acetylation site of RPA1 renders the cell hypersensitive to UV irradiation. HeLa cells stably expressing HA-Flag-tagged wild-type RPA1 or the K163R mutant were infected with lentiviral shRNA constructs that target the 3′ UTR of RPA1 and cultured in medium containing puromycin for 2 days. The resulting cells were treated with the indicated doses of UV, CPT, or HU. Cells were then permitted to grow for 14 days before staining. Experiments were performed in triplicates. Results shown are means of three independent experiments and were presented as means ± SEM. ∗∗p < 0.01; ∗∗∗p < 0.001. (E) RPA1 acetylation is not required for UV-induced PCNA monoubiquitylation. HeLa cells stably expressing HA-Flag-tagged wild-type RPA1 or the K163R mutant were infected with lentiviral shRNA constructs that target the 3′ UTR of RPA1 and cultured in medium containing puromycin for 2 days. The resulting cells were either mock treated or treated with 40 J/m2 UV and allowed to recover for 4 hr. The levels of the indicated proteins were analyzed by western blot. (F and G) RPA1 acetylation is not required for Polη foci formation. HeLa cells stably expressing HA-Flag-tagged wild-type RPA1 or the K163R mutant were infected with lentiviral shRNA constructs that target the 3′ UTR of RPA1 and cultured in medium containing puromycin for 2 days. The resulting cells were transfected with GFP- Polη and treated with 40 J/m2 UV. 4 hr later, cells were fixed and processed for Polη immunofluorescence. Representative Polη foci were shown (F). Quantification results were the average of three independent experiments and were presented as means ± SEM (G). More than one hundred cells were counted in each experiment. Cell Reports 2017 20, 2010-2025DOI: (10.1016/j.celrep.2017.08.016) Copyright © 2017 The Authors Terms and Conditions

Figure 6 RPA1 Acetylation Promotes NER (A–C) RPA1 acetylation is required for the efficient removal of UV-induced DNA lesions. HeLa cells stably expressing HA-Flag-tagged wild-type RPA1 or the K163R mutant were infected with lentiviral shRNA constructs that target the 3′ UTR of RPA1 and cultured in medium containing puromycin for 2 days. The resulting cells were irradiated with UV through 5-μm filters and allowed to recover for the indicated times. Cells were then stained with antibody for CPD (A) or 6-4PP (B). Results shown are means of three independent experiments and were presented as means ± SEM (C). More than 500 CPD or 6-4PP spots were counted for each condition. (D) RPA1 acetylation enhances its interaction with XPA. HeLa cells stably expressing HA-Flag-tagged wild-type RPA1, the K163R mutant, or the K163Q mutant were infected with lentiviral shRNA constructs that target the 3′ UTR of RPA1 and cultured in medium containing puromycin for 2 days. Cells were then mock treated or treated with 40 J/m2 UV and allowed to recover for 4 hr. Cell lysates were incubated with protein A agarose beads conjugated with anti-Flag antibody, and western blot analysis was performed with indicated antibodies. (E) Inhibition of GCN5 decreases the interaction between RPA1 and XPA. HeLa cells were either mock treated or treated with GCN5 inhibitor MB-3 (50 μM). 11 hr later, cells were then treated with 40 J/m2 UV and allowed to recover for 4 hr. Cell lysates were incubated with protein A agarose beads conjugated with anti-RPA1 antibody, and western blot analysis was performed with indicated antibodies. (F) Inhibition of HDAC6 enhances the interaction between RPA1 and XPA. HeLa cells were either mock treated or treated with HDAC6 inhibitor tubacin (20 μM) for 6 hr. Cell lysates were incubated with protein A agarose beads conjugated with anti-RPA1 antibody, and western blot analysis was performed with indicated antibodies. (G and H) RPA1 acetylation is required for XPA retention at sites of UV damage. HeLa cells stably expressing HA-Flag-tagged wild-type RPA1 or the K163R mutant were infected with lentiviral shRNA constructs that target the 3′ UTR of RPA1 and cultured in medium containing puromycin for 2 days. The resulting cells were irradiated with UV through 5-μm filters and allowed to recover for the indicated times. Cells were then stained with antibodies for XPA and RPA1 (α-HA; H). Quantification of the percentage of cells with RPA1-positive spots that are also positive for XPA is shown (G). Results shown are means of three independent experiments and were presented as means ± SEM. More than 200 RPA1-positive spots were counted for each condition. Cell Reports 2017 20, 2010-2025DOI: (10.1016/j.celrep.2017.08.016) Copyright © 2017 The Authors Terms and Conditions

Figure 7 RPA1 Acetylation Is Not Required for UV-Induced Damage Checkpoint Activation (A) RPA1 acetylation does not affect its ssDNA-binding ability. ssDNA-containing streptavidin beads were incubated with lysates prepared from RPA1-depleted HeLa cells stably expressing either wild-type RPA1 or the K163R mutant at 4°C for 1.5 hr. After washing, the samples were boiled with 2× SDS loading buffer, and western blot analysis was performed with indicated antibodies. (B) Mutation of the acetylation site of RPA1 does not affect its interaction with ATRIP. HeLa cells stably expressing HA-Flag-tagged wild-type RPA1 or the K163R mutant were infected with lentiviral shRNA constructs that target the 3′ UTR of RPA1 and cultured in medium containing puromycin for 2 days. The resulting cells were treated with 40 J/m2 UV and allowed to recover for 4 hr. Cell lysates were immunoprecipitated with anti-Flag antibody, and western blot analysis was performed with indicated antibodies. (C) RPA1 acetylation is not required for ATRIP recruitment to UV-induced DNA damage sites. HeLa cells stably expressing HA-Flag-tagged wild-type RPA1 or the K163R mutant were infected with lentiviral shRNA constructs that target the 3′ UTR of RPA1 and cultured in medium containing puromycin for 2 days. The resulting cells were irradiated with UV through 5-μm filters and allowed to recover for 4 hr. Cells were then stained with antibodies for ATRIP and Flag. (D) RPA1 acetylation is not required for UV-induced CHK1 phosphorylation. HeLa cells stably expressing HA-Flag-tagged wild-type RPA1 or the K163R mutant were infected with lentiviral shRNA constructs that target the 3′ UTR of RPA1 and cultured in medium containing puromycin for 2 days. The resulting cells were treated with 40 J/m2 UV and allowed to recover for the indicated times. Western blot analysis was performed with indicated antibodies. (E) A model of the role for RPA1 acetylation in the NER pathway. Cell Reports 2017 20, 2010-2025DOI: (10.1016/j.celrep.2017.08.016) Copyright © 2017 The Authors Terms and Conditions