SUMOylation of XRCC1 activated by poly (ADP-ribosyl)ation regulates DNA repair

SUMOylation of XRCC1 activated by poly (ADP-ribosyl)ation regulates DNA repair Abstract XRCC1 is an essential scaffold protein for base excision repair (BER) and helps to maintain genomic stability. XRCC1 has been indicated as a substrate for small ubiquitin-like modifier modification (SUMOylation); however, how XRCC1 SUMOylation is regulated in cells and how SUMOylated XRCC1 regulates BER activity are not well understood. Here, we show that SUMOylation of XRCC1 is regulated in cells under methyl-methanesulfonate (MMS) treatment and facilitates BER. Poly(ADP-ribose) polymerase 1 (PARP1) is activated by MMS immediately and synthesizes poly(ADP-ribose) (PAR), which in turn promotes recruitment of SUMO E3 TOPORS to XRCC1 and facilitates XRCC1 SUMOylation. A SUMOylation-defective mutant of XRCC1 had lower binding activity for DNA polymerase beta (POLB) and was linked to a lower capacity for repair of MMS-induced DNA damages. Our study therefore identified a pathway in which DNA damage-induced poly(ADP-ribosyl)ation (PARylation) promotes SUMOylation of XRCC1, which leads to more efficient recruitment of POLB to complete BER. Introduction Base excision repair (BER) fixes DNA base lesions and single-strand breaks (SSBs), which are caused by ubiquitous endogenous and exogenous agents, including reactive oxygen species, alkylating agents, and ionizing irradiation. Impaired BER results in both a decreased cellular capacity to counter genotoxic stress and increased genomic instability. BER is initiated by excision of the damaged base by DNA glycosylases and subsequently leads to a SSB, which is then filled by DNA polymerase β (POLB) and DNA ligase III (LIG3) (1). When SSBs are formed, the DNA-damage response (DDR) is activated to promote efficient BER via phosphorylation of the BER proteins by ATM/ATR-associated signals (2) and poly(ADP-ribosyl)ation (PARylation) of multiple BER proteins by poly(ADP-ribose) polymerases (PARPs) (3). XRCC1 plays a central role in BER, and its loss in cells leads to increased chromosomal instability and impaired DNA repair (4). Interestingly, XRCC1 itself has no enzymatic activity, but the scaffold function of XRCC1 is essential for BER (4,5). XRCC1 specifically interacts with other BER components through different domains: the N-terminal domain (NTD) interacts with POLB (6), the BRCA1 C-terminal (BRCT) I domain interacts with PARP (7), and the BRCT II domain interacts with LIG3 (8). Therefore, we wished to identify the regulatory mechanisms that control XRCC1 interactions with other BER components. Post-translational modification (PTM) of proteins by PARylation, phosphorylation, ubiquitination, and small ubiquitin-like modifier modification (SUMOylation) is a well-recognized mechanism that dynamically regulates the cellular functions of proteins, including not only the cell cycle and apoptosis but also the DDR and DNA repair (9). In particular, the formation of the repair complex is influenced by DNA damage–induced PTMs, which increases the specificity and efficiency of BER (10). Several PTMs were recently identified as mechanisms involved in the regulation of BER proteins in response to DNA damage (10). PTM of XRCC1 for DDR and DNA repair occurs via phosphorylation, ubiquitination, and PARylation. For example, phosphorylation at multiple sites within XRCC1 by protein kinases CK2 and Chk2 is required to maintain its stability and promote efficient DNA repair, respectively (11–13). XRCC1 is also ubiquitinated by the E3 ligase CHIP during the steady state (14) and by Iduna when DNA damage occurs (15); PARylation of XRCC1 prevents DNA damage-induced ubiquitylation (16). These observations suggest the importance of multiple PTMs of XRCC1 during BER. Protein SUMOylation has recently been suggested as a PTM that is critical for DDR and DNA repair (9). The SUMO proteins, SUMO-1, -2, and -3, share structural similarities with ubiquitin and form isopeptide linkages with specific lysine residues within the consensus motif Ψ-K-x-D/E (Ψ is a hydrophobic amino acid and x is any amino acid residue) in their substrates through an enzymatic cascade involving the sequential action of E1 activator, E2 conjugase, and E3 ligase (17). SUMO proteins are proposed to regulate the function of DNA repair proteins, including thymine-DNA glycosylase (18), BRCA1 (19), BLM (20), RPA70 (21), and MDC1 (22). Because protein SUMOylation is important in DNA repair and because PTM of XRCC1 is critical for its function in BER, we hypothesized that XRCC1 may increase SUMOylation upon DNA damage and that this PTM may be involved in the regulation of XRCC1 function in BER via the impact on repair complex formation and DNA repair efficiency. In this study, we found that the DNA alkylating-agent methyl methanesulfonate (MMS) induces PARP1 activation and PAR synthesis, and subsequently recruits SUMO E3 TOPORS to the XRCC1 complex. Furthermore, the interaction between TOPORS and XRCC1 facilitates XRCC1 SUMOylation, which leads to more-efficient BER via its ability to efficiently bind with POLB and thus contributes to cell survival after MMS-induced damage. Results DNA alkylating-agent MMS increases XRCC1 SUMOylation Although XRCC1 was reported to be SUMOylated both in vitro and in vivo (23–26), it is unclear how XRCC1 SUMOylation is regulated in cells. To understand this, we explored whether endogenous XRCC1 SUMOylation can be regulated in cells treated with MMS. Immunoblotting (IB) analysis of immunoprecipitated endogenous XRCC1 from 293T cells revealed that one major and one minor slower-migrating bands of XRCC1 were increased by MMS treatment (Fig. 1A, right, asterisk and double asterisks, respectively). The bands were also detected by anti-SUMO1 and anti-SUMO2/3 antibodies (Fig. 1B and C). The endogenous XRCC1 SUMOylation increased by MMS was also demonstrated by IP-IB analysis in breast cancer cell line MCF-7 (Supplementary Material, Fig. S1). Similar results were obtained with overexpression of FLAG-XRCC1 and EGFP-SUMO1 (Fig. 1D) or EGFP-SUMO2 (Fig. 1E). We further demonstrated that these two bands were SUMOylated XRCC1 because UBC9 knockdown notably reduced the slowly migrating XRCC1 bands (Fig. 1F). These results suggested that MMS treatment upregulated XRCC1 SUMOylation. Figure 1. View largeDownload slide  SUMOylation of XRCC1 was increased in response to alkylating DNA damage. (A–C) SUMOylated XRCC1 was increased by MMS treatment. 293T cells were treated with MMS (0.3 mg/ml for 1 h), and cell lysates were prepared and immunoprecipitated with anti-XRCC1 and immunoblotted with anti-XRCC1 (A), anti-SUMO1 (B), or anti-SUMO2/3 (C). Left panel in (A), immunoblots show 2.5% input of each lysate subjected to the immunoprecipitation (IP) assay with the indicated antibodies. Normal IgG was used as a negative control for IP assays. The ratio of SUMO-modified/unmodified XRCC1 band was indicated under the blots. (D and E) Immunoblots show MMS increased SUMOylation of XRCC1 in 293T cells that overexpressed FLAG-XRCC1 and EGFP-SUMO1 (D) or EGFP-SUMO2 (E). (F) Immunoblots show that SUMOylation of XRCC1 was reduced by siRNA-mediated knockdown of UBC9 in 293T cells with (+) and without (C) MMS treatment. (G) An in vitro SUMOylation assay was performed with the recombinant protein N-terminal GST-tagged and C-terminal His-tagged XRCC1, His-tagged SUMO1, and SUMOylation components. The in vitro SUMOylation reaction was performed and analyzed by immunoblotting with anti-His antibody. (H) Immunoblots show that the consensus SUMO motif VKEE in XRCC1 is the major acceptor for SUMOylation. SUMOylation of XRCC1 was abolished in 293T cells co-transfected with FLAG-XRCC1 mutant K176R or E178A along with EGFP-SUMO1. The SUMOylated XRCC1 bands were analyzed by immunoblotting (IB) with anti-FLAG and anti-GFP antibodies. In immunoblots here and throughout the figures, an arrowhead indicates unmodified XRCC1, an asterisk (*) indicates the major SUMO-modified XRCC1 band, double asterisks (**) indicate the minor SUMO-modified XRCC1 band, an arrow indicates a non-specific band, numbers to the side of the blots indicate molecular weight in kilodalton (kDa)s, anti-Tubulin or anti-Lamin B are used as loading control, MMS-induced DNA damage is indicated with anti-Chk2 (phospho-T68) or anti-p53 (phospho-S15), and C or Control indicates that dimethyl sulfoxide (DMSO) was used as the vehicle control. Figure 1. View largeDownload slide  SUMOylation of XRCC1 was increased in response to alkylating DNA damage. (A–C) SUMOylated XRCC1 was increased by MMS treatment. 293T cells were treated with MMS (0.3 mg/ml for 1 h), and cell lysates were prepared and immunoprecipitated with anti-XRCC1 and immunoblotted with anti-XRCC1 (A), anti-SUMO1 (B), or anti-SUMO2/3 (C). Left panel in (A), immunoblots show 2.5% input of each lysate subjected to the immunoprecipitation (IP) assay with the indicated antibodies. Normal IgG was used as a negative control for IP assays. The ratio of SUMO-modified/unmodified XRCC1 band was indicated under the blots. (D and E) Immunoblots show MMS increased SUMOylation of XRCC1 in 293T cells that overexpressed FLAG-XRCC1 and EGFP-SUMO1 (D) or EGFP-SUMO2 (E). (F) Immunoblots show that SUMOylation of XRCC1 was reduced by siRNA-mediated knockdown of UBC9 in 293T cells with (+) and without (C) MMS treatment. (G) An in vitro SUMOylation assay was performed with the recombinant protein N-terminal GST-tagged and C-terminal His-tagged XRCC1, His-tagged SUMO1, and SUMOylation components. The in vitro SUMOylation reaction was performed and analyzed by immunoblotting with anti-His antibody. (H) Immunoblots show that the consensus SUMO motif VKEE in XRCC1 is the major acceptor for SUMOylation. SUMOylation of XRCC1 was abolished in 293T cells co-transfected with FLAG-XRCC1 mutant K176R or E178A along with EGFP-SUMO1. The SUMOylated XRCC1 bands were analyzed by immunoblotting (IB) with anti-FLAG and anti-GFP antibodies. In immunoblots here and throughout the figures, an arrowhead indicates unmodified XRCC1, an asterisk (*) indicates the major SUMO-modified XRCC1 band, double asterisks (**) indicate the minor SUMO-modified XRCC1 band, an arrow indicates a non-specific band, numbers to the side of the blots indicate molecular weight in kilodalton (kDa)s, anti-Tubulin or anti-Lamin B are used as loading control, MMS-induced DNA damage is indicated with anti-Chk2 (phospho-T68) or anti-p53 (phospho-S15), and C or Control indicates that dimethyl sulfoxide (DMSO) was used as the vehicle control. We next delineated the XRCC1 SUMOylation site(s). We performed an in vitro SUMOylation assay with XRCC1 recombinant protein. The results also showed one major and one minor SUMOylated XRCC1 band (Fig. 1G and Supplementary Material, Fig. S2). The major SUMOylated XRCC1 band was further subjected to mass spectrometry analysis and revealed K176 as a SUMOylation site (Supplementary Material, Fig. S2C), consistent with a report showing that XRCC1 was SUMOylated by SUMO2 (25) and the XRCC1 K176R mutant was defective in SUMOylation based on an Escherichia coli SUMOylation system (26). We further confirmed K176 as the major SUMOylation site in cells with MMS treatment (Fig. 1H). This XRCC1 SUMOylation site, VKEEDE, is consistent with the SUMOylation consensus motif Ψ-K-x-D/E (27). Mutation of acidic residue E within this motif should reduce the XRCC1 SUMOylation. As expected, the E178A mutant decreased XRCC1 SUMOylation in cells (Fig. 1H). Note that the minor SUMOylated band of XRCC1 was not affected by the K176R or E178A mutation. MMS-induced SUMOylation of XRCC1 is regulated by PARylation To provide a more comprehensive view, we investigated whether the MMS-induced DDR signaling is involved in the regulation of XRCC1 SUMOylation. PARylation, an immediately-early reaction of DDR, is activated by PARPs in response to genotoxic stress, and after completing its effect, the degradation of poly-(ADP-ribose) (PAR) chain is regulated by the opposing reaction of PARG (28). If, indeed, XRCC1 SUMOylation is regulated by PARylation, it would be expected that the level of XRCC1 SUMOylation would decrease in cells with suppressed PARP activity or would increase with suppressed PARG activity. Thus, we manipulated the activity of PARP and PARG in 293T cells to provide evidence for the involvement of PARylation in XRCC1 SUMOylation. Indeed, SUMOylation of XRCC1 was decreased when PARP activity was inhibited by the PARP-specific inhibitor 4-amino-1,8-naphthalimide (4-AN) and increased in cells treated with PARG inhibitor tannic acid (Fig. 2A). Given that PARP1 is the most important member of PARP family for the synthesis of PAR chains (29), we demonstrated that siRNA knockdown of endogenous PARP1 reduced MMS-induced XRCC1 SUMOylation (Fig. 2B); whereas knockdown of PARG increased XRCC1 SUMOylation (Fig. 2C). Consistent to this expectation, the endogenous level of MMS-induced XRCC1 SUMOylation was decreased by PARP inhibitor 4-AN (Fig. 2D). Taken together, these results demonstrated that MMS-induced SUMOylation of XRCC1 is regulated by PARylation, which is mainly catalyzed by PARP1. Figure 2. View largeDownload slide  MMS-induced SUMOylation of XRCC1 is regulated by PARylation. (A) Immunoblots show that SUMOylation of XRCC1 is modulated by a PARylation-related inhibitor. 293T cells that co-expressed FLAG-XRCC1 and EGFP-SUMO1 were treated with MMS after pre-treatment with a specific PARP inhibitor (4-AN, 10μM) or PARG inhibitor (tannic acid, 100 μM) for 1 h. (B) Immunoblots show that SUMOylation of XRCC1 was decreased by knockdown of PARP1 with a specific siRNA in 293T cells co-transfected with FLAG-XRCC1 and EGFP-SUMO1. (C) MMS-induced SUMOylation of XRCC1 was increased by depletion of PARG. Prior to MMS treatment, 293T cells were transfected with PARG-specific siRNA along with FLAG-XRCC1 and EGFP-SUMO1. The lysates from transfected cells were analyzed by IB with anti-GFP and anti-FLAG antibodies. The efficiency of PARG knockdown was quantified with real-time PCR and normalized to the expression of GAPDH. Data represent the mean ± SD (Student’s t-test.) from two experiments performed in triplicate. (D) Immunoblots of immunoprecipitated endogenous XRCC1 show that MMS-increased SUMOylated-XRCC1 was reduced by PARP inhibitor 4-AN. 293T cells were pretreatment with or without 4-AN (10 μM) for 1 h and then continued incubation following MMS for 1 h, and then cell lysates were subjected to IP with anti-XRCC1 antibody. These blots has been cropped and the full-length blots of IPs (D, right panel) were included in Supplementary Material. Figure 2. View largeDownload slide  MMS-induced SUMOylation of XRCC1 is regulated by PARylation. (A) Immunoblots show that SUMOylation of XRCC1 is modulated by a PARylation-related inhibitor. 293T cells that co-expressed FLAG-XRCC1 and EGFP-SUMO1 were treated with MMS after pre-treatment with a specific PARP inhibitor (4-AN, 10μM) or PARG inhibitor (tannic acid, 100 μM) for 1 h. (B) Immunoblots show that SUMOylation of XRCC1 was decreased by knockdown of PARP1 with a specific siRNA in 293T cells co-transfected with FLAG-XRCC1 and EGFP-SUMO1. (C) MMS-induced SUMOylation of XRCC1 was increased by depletion of PARG. Prior to MMS treatment, 293T cells were transfected with PARG-specific siRNA along with FLAG-XRCC1 and EGFP-SUMO1. The lysates from transfected cells were analyzed by IB with anti-GFP and anti-FLAG antibodies. The efficiency of PARG knockdown was quantified with real-time PCR and normalized to the expression of GAPDH. Data represent the mean ± SD (Student’s t-test.) from two experiments performed in triplicate. (D) Immunoblots of immunoprecipitated endogenous XRCC1 show that MMS-increased SUMOylated-XRCC1 was reduced by PARP inhibitor 4-AN. 293T cells were pretreatment with or without 4-AN (10 μM) for 1 h and then continued incubation following MMS for 1 h, and then cell lysates were subjected to IP with anti-XRCC1 antibody. These blots has been cropped and the full-length blots of IPs (D, right panel) were included in Supplementary Material. PAR-mediated SUMO E3 ligase TOPORS recruitment to XRCC1 promotes SUMOylation To explore in-depth mechanisms of the role of PARylation in XRCC1 SUMOylation, we investigated how PARylation facilitates XRCC1 SUMOylation. Upon MMS treatment, PARP-mediated PARylation is the DDR that helps to recruit proteins to sites of DNA damage to promote efficient BER (30). PAR can serve as a molecular matrix to promote protein–protein interactions (28,31). Protein PARylation has been suggested to regulate SUMOylation of IKKgamma by recruitment of SUMO E3 ligase PIASy in cells under genotoxic stress (32). Given our finding above that XRCC1 is a SUMOylated target, the next question would be which SUMO E3 ligases regulate SUMOylation of XRCC1 and whether PAR is involved in the interaction between XRCC1 and E3 ligase. The SUMO E3 ligases, including TOPORS (33,34), PIAS1 (35), CBX4 (also referred to as Pc2) (36), and MMS21 (37), have been reported to be involved in responses to DNA damage. We examined whether the protein level of these E3 ligases is notably altered after MMS treatment. Importantly, the endogenous level of TOPORS, but not of PIAS1, CBX4, and MMS21, was gradually increased by MMS treatment and returned to baseline levels after 2 h (Fig. 3A). We then performed knockdown experiments and found TOPORS to be the most important SUMO E3 ligase for XRCC1 (Fig. 3B). Additionally, we confirmed that the increase in TOPORS level upon MMS treatment was not due to regulation at the transcriptional level, as assessed by real-time qPCR (Supplementary Material, Fig. S3). These results indicated that SUMOylation of XRCC1 was correlated with the protein level of TOPORS and led us to suggest TOPORS is a SUMO E3 ligase for XRCC1. Figure 3. View largeDownload slide  PAR-mediated SUMO E3 ligase TOPORS recruitment to XRCC1 to promote SUMOylation. (A) Immunoblots show that the level of the SUMO E3 ligase TOPORS was obviously altered by MMS treatment in this time-course experiment, but not the level of CBX4, PIAS1, or MMS21. (B) Immunoblots show that the level of SUMOylated XRCC1 was decreased by knockdown of TOPORS with a specific siRNA, but not by knockdown of CBX4 or MMS21. (C) Endogenous XRCC1 and TOPORS were co-immunoprecipitated by anti-PAR antibody after MMS treatment. 293T cells were treated with MMS for indicated time periods in the absence or in the presence of 4-AN (pretreatment with 4-AN, 10 μM for 1 h), and then cell lysates were subjected to IP with anti-PAR antibody. Proteins in the immunoprecipitated complex were analyzed by IB with anti-PAR, anti-XRCC1, and anti-TOPORS antibodies. (D) Immunoblots show that the association of TOPORS with XRCC1 increased after MMS treatment in 293T cells transfected with FLAG-XRCC1 and Myc-TOPORS. (E) Immunoblots show that both PAR and TOPORS had increased association with XRCC1 upon treatment with MMS in 293T cells transfected with FLAG-XRCC1 and Myc-TOPORS, and then cell lysates were subjected to IP with anti-FLAG antibody. The association between TOPORS and XRCC1 was reduced by PARP inhibitor 4-AN. The blots were cropped and the full-length blots of IPs (E) were included in Supplementary Material. Figure 3. View largeDownload slide  PAR-mediated SUMO E3 ligase TOPORS recruitment to XRCC1 to promote SUMOylation. (A) Immunoblots show that the level of the SUMO E3 ligase TOPORS was obviously altered by MMS treatment in this time-course experiment, but not the level of CBX4, PIAS1, or MMS21. (B) Immunoblots show that the level of SUMOylated XRCC1 was decreased by knockdown of TOPORS with a specific siRNA, but not by knockdown of CBX4 or MMS21. (C) Endogenous XRCC1 and TOPORS were co-immunoprecipitated by anti-PAR antibody after MMS treatment. 293T cells were treated with MMS for indicated time periods in the absence or in the presence of 4-AN (pretreatment with 4-AN, 10 μM for 1 h), and then cell lysates were subjected to IP with anti-PAR antibody. Proteins in the immunoprecipitated complex were analyzed by IB with anti-PAR, anti-XRCC1, and anti-TOPORS antibodies. (D) Immunoblots show that the association of TOPORS with XRCC1 increased after MMS treatment in 293T cells transfected with FLAG-XRCC1 and Myc-TOPORS. (E) Immunoblots show that both PAR and TOPORS had increased association with XRCC1 upon treatment with MMS in 293T cells transfected with FLAG-XRCC1 and Myc-TOPORS, and then cell lysates were subjected to IP with anti-FLAG antibody. The association between TOPORS and XRCC1 was reduced by PARP inhibitor 4-AN. The blots were cropped and the full-length blots of IPs (E) were included in Supplementary Material. Based on the data mentioned earlier, we hypothesized that PAR is involved in the MMS-induced interaction between the SUMO E3 ligase TOPORS and XRCC1. To examine this, we assessed the binding of PAR with XRCC1 and TOPORS in 293T cells after MMS treatment. IB analysis of immunoprecipitated endogenous PAR revealed that XRCC1 and TOPORS were both present in the PAR complex, and their levels in the complex increased upon MMS treatment (Fig. 3C). These results also demonstrated in breast cancer cell lines MCF-7 and MDA-MB-453 cells (Supplementary Material, Fig. S4A and B). We further demonstrated that MMS treatment also increased the interaction between Myc-TOPORS and FLAG-XRCC1 as the result of a co-immunoprecipitation (co-IP) assay with anti-FLAG (Fig. 3D). Consistently, the recruitment of TOPORS to XRCC1 via PAR induced by MMS treatment was suppressed by the PARP inhibitor 4-AN and, as a result, the SUMOylation of XRCC1 was decreased by 4-AN in 293T cells that co-expressed FLAG-XRCC1 and Myc-TOPORS (Fig. 3E). These results led us to suggest that MMS-induced XRCC1 SUMOylation is regulated by PAR through the recruitment of SUMO E3 ligase TOPORS to XRCC1 to promote SUMOylation. PAR-binding of XRCC1 is crucial for MMS-induced XRCC1 SUMOylation We showed above that PAR mediated the formation of XRCC1 and TOPORS complex. Thus, in the following experiments, we examined the PAR-binding ability of XRCC1 and TOPORS is important for the association of XRCC1 and TOPORS. The C-terminus of TOPORS is required for SUMOylation via interaction with SUMO1 and UBC9 (38) and is a potential PAR-binding segment containing an RS domain (SR-rich domain), i.e. a potential PAR-binding domain (28) (Supplementary Material, Fig. S5A). Thus, we performed specific experiments and demonstrated that the C-terminus of TOPORS is required for its binding ability to XRCC1 and PAR (Supplementary Material, Fig. S5B–E), and is also required for XRCC1 SUMOylation in vitro (Supplementary Material, Fig. S6). Furthermore, the co-IP results showed that the level of MMS-induced XRCC1 SUMOylation and the interaction of XRCC1 and TOPORS-C were suppressed by the PARP inhibitor (Fig. 4A). XRCC1 binds to PAR through its BRCT I domain (31), and the PAR binding-defective XRCC1 mutant RK (R335A/K369A) reduces DNA repair activity and cell survival after MMS-induced damage (39). Thus, to clarify the involvement of PAR in promoting XRCC1 SUMOylation, biotin-PAR or in vitro PARylated-PARP1 was pre-incubated with recombinant GST-tagged XRCC1 protein, followed by an in vitro SUMOylation assay with purified HA-tagged TOPORS-C and SUMOylation components (Fig. 4B, left). Immunoblots showed SUMOylation of XRCC1 was increased by pre-incubation with either biotin-PAR or PARylated-PARP1, but not with PARP1 alone in the absence of NAD+ (Fig. 4B, right). To further confirm whether the PAR-binding activity of XRCC1 is required for SUMOylation, we examined the SUMOylation of XRCC1 in 293T cells that expressed FLAG-tagged WT or RK XRCC1 with EGFP-SUMO1. The PAR binding-defective mutant of XRCC1 attenuated MMS-induced XRCC1 SUMOylation (Fig. 4C) and reduced its interaction with PAR and TOPORS-C (Fig. 4D). We further demonstrated that XRCC1 SUMOylation was promoted by overexpression of full-length TOPORS. In contrast, the level of XRCC1 SUMOylation and the binding of XRCC1 to TOPORS were reduced in XRCC1 PAR binding-defective mutant (Fig. 4E). Taken together, these data provide additional support for the involvement of PARylation in promoting SUMOylation of XRCC1 by facilitating the formation of the XRCC1/TOPORS complex upon MMS treatment. Figure 4. View largeDownload slide  PAR-binding of XRCC1 is crucial for MMS-induced XRCC1 SUMOylation. (A) Immunoblots show that the association between XRCC1 and TOPORS-C decreased upon treatment with PARP inhibitor 4-AN. 293T cells transfected with HA-TOPORS-C and FLAG-XRCC1 were pre-treated with 4-AN for 1 h before treatment with MMS, and then cell lysates were subjected to IP with anti-FLAG antibody. (B) SUMOylation of XRCC1 was increased by addition of biotin-PAR and by pre-PARylated PARP1 in a cell-free system. GST-tagged XRCC1-His protein bound to GST beads was incubated with biotin-PAR or the PARylation product of PARP1. After washing, an in vitro SUMOylation assay was performed in the presence of purified HA-tagged TOPORS-C protein. The reaction was analyzed by IB with anti-SUMO1 and anti-GST antibodies. (C) Immunoblots show that SUMOylation of RK XRCC1 was decreased in 293T cells that co-expressed EGFP-SUMO1 with FLAG-tagged WT or RK XRCC1 after MMS treatment for 1 h. (D) Immunoblots show that the ability of XRCC1 to bind to TOPORS-C was decreased in the RK XRCC1 PAR-binding mutant. 293T cells transfected with HA-TOPORS-C and FLAG-XRCC1 were treated with MMS for 30 min, and then cell lysates were subjected to IP with anti-FLAG antibody. (E) Immunoblots show that the SUMOylation of XRCC1 was increased by overexpression of full-length TOPORS and the ability of XRCC1 to bind to TOPORS was higher in WT than in the RK XRCC1 PAR-binding mutant. 293T cells transfected with Myc-TOPORS, EGFP-SUMO1, and FLAG-XRCC1 were treated with MMS for 1 h, and then cell lysates were subjected to IP with anti-FLAG antibody. These blots has been cropped. Figure 4. View largeDownload slide  PAR-binding of XRCC1 is crucial for MMS-induced XRCC1 SUMOylation. (A) Immunoblots show that the association between XRCC1 and TOPORS-C decreased upon treatment with PARP inhibitor 4-AN. 293T cells transfected with HA-TOPORS-C and FLAG-XRCC1 were pre-treated with 4-AN for 1 h before treatment with MMS, and then cell lysates were subjected to IP with anti-FLAG antibody. (B) SUMOylation of XRCC1 was increased by addition of biotin-PAR and by pre-PARylated PARP1 in a cell-free system. GST-tagged XRCC1-His protein bound to GST beads was incubated with biotin-PAR or the PARylation product of PARP1. After washing, an in vitro SUMOylation assay was performed in the presence of purified HA-tagged TOPORS-C protein. The reaction was analyzed by IB with anti-SUMO1 and anti-GST antibodies. (C) Immunoblots show that SUMOylation of RK XRCC1 was decreased in 293T cells that co-expressed EGFP-SUMO1 with FLAG-tagged WT or RK XRCC1 after MMS treatment for 1 h. (D) Immunoblots show that the ability of XRCC1 to bind to TOPORS-C was decreased in the RK XRCC1 PAR-binding mutant. 293T cells transfected with HA-TOPORS-C and FLAG-XRCC1 were treated with MMS for 30 min, and then cell lysates were subjected to IP with anti-FLAG antibody. (E) Immunoblots show that the SUMOylation of XRCC1 was increased by overexpression of full-length TOPORS and the ability of XRCC1 to bind to TOPORS was higher in WT than in the RK XRCC1 PAR-binding mutant. 293T cells transfected with Myc-TOPORS, EGFP-SUMO1, and FLAG-XRCC1 were treated with MMS for 1 h, and then cell lysates were subjected to IP with anti-FLAG antibody. These blots has been cropped. SUMOylation of XRCC1 promotes efficient DNA repair and cell survival after MMS treatment XRCC1 is required for DNA repair and survival from toxic DNA lesions induced by MMS (4). After the understanding of SUMOylation of XRCC1 upon MMS treatment, we investigated whether SUMOylation affects the function of XRCC1 in repairing MMS-induced DNA damages and cell survival. To this end, we first established CHO-derived XRCC1-deficient EM9 cells that stably expressed either FLAG-tagged XRCC1 WT or the K176R mutant (Fig. 5A) and then assessed the MMS-induced base damage repair using the comet assay. Compared with XRCC1 WT, XRCC1 K176R significantly reduced DNA repair, as evidenced by retained tail DNA (Fig. 5B and Supplementary Material, Fig. S7A) and consequently reduced clonogenic cell survival (Fig. 5C and Supplementary Material, Fig. S7B). Similar result was also demonstrated in transiently transfected MDA-MB-453 breast cancer cells (Supplementary Material, Fig. S8). Furthermore, we showed that the difference in survival between MMS-treated XRCC1 WT and K176R mutant stable cells was decreased if cells were pre-treated with PARP inhibitor 4-AN (Fig. 5D). These data demonstrated that SUMOylation of XRCC1 facilitates DNA repair and survival, and further supports for the suggestion that XRCC1 SUMOylation in BER is mediated by MMS-activated PARylation. Figure 5. View largeDownload slide  SUMOylation of XRCC1 enhances DNA repair and prevents cell death. (A) Immunoblots show the stable expression of XRCC1 WT or K176R in CHO-derived XRCC1-deficient EM9 cells. Cells expressing the empty vector were used as a control. (B) Quantification of the MMS-induced damaged DNA (comet tail) shows that EM9 cells harboring K176R XRCC1 retained more damaged DNA after recovery. Cells were treated with MMS (0.1 mg/ml) for 15 min, and then allowed to recover for 1 h. The data represent the % of DNA in comet tail from three independent experiments. For each treatment, 80∼100 cells were quantified. The results were analyzed by Prism. ***, P < 0.001 based on t-test. (C) Cells harboring K176R XRCC1 were more sensitive to MMS treatment. EM9-derived XRCC1 WT or K176R stable cells were treated with various doses of MMS for 15 min and then examined for colony-forming ability. Survival is expressed as a percentage of the untreated control for each clone (mean ± SD) from three independent experiments performed in triplicate. The generalized linear model (GLM) was used to test for a linear relationship (trend) of WT and K176R XRCC1 cells after serial doses of MMS treatment. ***, P < 0.05 was the limit of significance. (D). The difference in MMS-induced cell death between WT and K176R XRCC1 cells was reduced by PARP inhibitor 4-AN. WT or K176R XRCC1 cells were incubated with various doses of 4-AN for 30 min and then were treated with MMS (0.15 mg/ml for 15 min) or a vehicle control. Cellular viability was measured with the MTT assay and is expressed as a percentage of the untreated control for each clone. Data are presented relative to untreated cells and represent the mean ± SD for one experiment performed in triplicate. These blots has been cropped. Figure 5. View largeDownload slide  SUMOylation of XRCC1 enhances DNA repair and prevents cell death. (A) Immunoblots show the stable expression of XRCC1 WT or K176R in CHO-derived XRCC1-deficient EM9 cells. Cells expressing the empty vector were used as a control. (B) Quantification of the MMS-induced damaged DNA (comet tail) shows that EM9 cells harboring K176R XRCC1 retained more damaged DNA after recovery. Cells were treated with MMS (0.1 mg/ml) for 15 min, and then allowed to recover for 1 h. The data represent the % of DNA in comet tail from three independent experiments. For each treatment, 80∼100 cells were quantified. The results were analyzed by Prism. ***, P < 0.001 based on t-test. (C) Cells harboring K176R XRCC1 were more sensitive to MMS treatment. EM9-derived XRCC1 WT or K176R stable cells were treated with various doses of MMS for 15 min and then examined for colony-forming ability. Survival is expressed as a percentage of the untreated control for each clone (mean ± SD) from three independent experiments performed in triplicate. The generalized linear model (GLM) was used to test for a linear relationship (trend) of WT and K176R XRCC1 cells after serial doses of MMS treatment. ***, P < 0.05 was the limit of significance. (D). The difference in MMS-induced cell death between WT and K176R XRCC1 cells was reduced by PARP inhibitor 4-AN. WT or K176R XRCC1 cells were incubated with various doses of 4-AN for 30 min and then were treated with MMS (0.15 mg/ml for 15 min) or a vehicle control. Cellular viability was measured with the MTT assay and is expressed as a percentage of the untreated control for each clone. Data are presented relative to untreated cells and represent the mean ± SD for one experiment performed in triplicate. These blots has been cropped. SUMOylation of XRCC1 facilitates DNA repair mediated through efficient POLB recruitment To further understand the XRCC1 SUMOylation-mediated-DNA repair, we provided supporting evidence by directly assessing the repair activity of specific BER members using cell extracts from these cell lines using the in vitro BER/SSBR assay (Fig. 6A). Similar to the observations with the comet assay, BER activity was significantly decreased in stable cells expressing the K176R mutant (Fig. 6B). The results implied that SUMOylation regulates the function of XRCC1 in BER. XRCC1 acts as a scaffold to recruit BER enzymes, including POLB and LIG3, to the sites of DNA damage, which is important for efficient damage repair (7). We hypothesized that SUMOylation may regulate the interaction of XRCC1 with BER repair proteins. To address this possibility, we assessed the binding of repair proteins POLB and LIG3 to WT and K176R XRCC1. The results of co-immunoprecipitation experiments showed that the XRCC1 K176R mutant notably decreased its interaction with POLB, but not LIG3 (Fig. 6C). Similar result was also demonstrated in MDA-MB-453 breast cancer cells (Supplementary Material, Fig. S9). Figure 6. View largeDownload slide SUMOylation of XRCC1 promotes BER activity mediated through efficient POLB recruitment. (A) Schematic representation of the process utilizing real-time PCR to quantify in vitro BER activity. Cell extract was incubated with synthesized template B (which includes a single-nucleotide gap) for the in vitro BER assay. Intact template C in the reaction was used as the input control. The repaired template B was quantified with TaqMan-based real-time PCR. (B) The XRCC1 K176R mutant had lower BER activity. The cell extract from EM9-derived XRCC1 WT or K176R stable cells was incubated with template B and control template C. Relative BER activity was normalized to the control (template C). Data are presented relative to WT XRCC1 (100%) and represent the mean ± SD for two independent experiments performed in triplicate. ***, P < 0.001 based on Student’s t-test. (C) The association of POLB with XRCC1 decreased in the SUMOylation-defective K176R mutant. IB of the protein complex was carried out after IP of XRCC1 with anti-FLAG antibody from stable cells. The lysate was analyzed in parallel. (D–F) SUMOylated XRCC1 pulled down more POLB protein and had a higher BER activity compared to unmodified XRCC1. Schematic representation of the process utilizing in vitro SUMOylated-XRCC1 to pull-down His-tagged POLB protein and further analyze with IB and an in vitro BER assays (D). Immunoblots show that SUMOylated XRCC1 pulled down more POLB protein. Recombinant POLB protein was incubated with SUMOylated GST-XRCC1-His protein and then GST-pulled down. The GST-pull down products P1 and P2 were analyzed by IB with anti-POLB antibody (E). The difference of BER activity in these two repair complex can be restored by supplement with extra recombinant POLB protein. The GST-pull down products P1 and P2 were subjected to BER assay supplement with recombinant POLB protein (F). (G and H). POLB preferentially binds to SUMOylated-XRCC1, as determined by far-western blotting (G) and immuno-pull-down (H) assays. For the far-western blotting, in vitro SUMOylated GST-XRCC1 was separated by SDS–PAGE, immobilized on a nitrocellulose membrane, and incubated with recombinant His-tagged POLB protein as bait, followed by IB with anti-POLB antibody (G, right). The input level of GST-XRCC1 was analyzed with anti-GST antibody (G, left). The ratio of modified/unmodified bands indicates the binding of POLB to SUMO-modified versus SUMO-unmodified XRCC1. For the pull-down assay, SUMOylated GST-XRCC1 was incubated with recombinant His-tagged POLB protein, followed by IP with anti-POLB antibody and IB with anti-GST and anti-POLB antibody (H). These blots has been cropped. Figure 6. View largeDownload slide SUMOylation of XRCC1 promotes BER activity mediated through efficient POLB recruitment. (A) Schematic representation of the process utilizing real-time PCR to quantify in vitro BER activity. Cell extract was incubated with synthesized template B (which includes a single-nucleotide gap) for the in vitro BER assay. Intact template C in the reaction was used as the input control. The repaired template B was quantified with TaqMan-based real-time PCR. (B) The XRCC1 K176R mutant had lower BER activity. The cell extract from EM9-derived XRCC1 WT or K176R stable cells was incubated with template B and control template C. Relative BER activity was normalized to the control (template C). Data are presented relative to WT XRCC1 (100%) and represent the mean ± SD for two independent experiments performed in triplicate. ***, P < 0.001 based on Student’s t-test. (C) The association of POLB with XRCC1 decreased in the SUMOylation-defective K176R mutant. IB of the protein complex was carried out after IP of XRCC1 with anti-FLAG antibody from stable cells. The lysate was analyzed in parallel. (D–F) SUMOylated XRCC1 pulled down more POLB protein and had a higher BER activity compared to unmodified XRCC1. Schematic representation of the process utilizing in vitro SUMOylated-XRCC1 to pull-down His-tagged POLB protein and further analyze with IB and an in vitro BER assays (D). Immunoblots show that SUMOylated XRCC1 pulled down more POLB protein. Recombinant POLB protein was incubated with SUMOylated GST-XRCC1-His protein and then GST-pulled down. The GST-pull down products P1 and P2 were analyzed by IB with anti-POLB antibody (E). The difference of BER activity in these two repair complex can be restored by supplement with extra recombinant POLB protein. The GST-pull down products P1 and P2 were subjected to BER assay supplement with recombinant POLB protein (F). (G and H). POLB preferentially binds to SUMOylated-XRCC1, as determined by far-western blotting (G) and immuno-pull-down (H) assays. For the far-western blotting, in vitro SUMOylated GST-XRCC1 was separated by SDS–PAGE, immobilized on a nitrocellulose membrane, and incubated with recombinant His-tagged POLB protein as bait, followed by IB with anti-POLB antibody (G, right). The input level of GST-XRCC1 was analyzed with anti-GST antibody (G, left). The ratio of modified/unmodified bands indicates the binding of POLB to SUMO-modified versus SUMO-unmodified XRCC1. For the pull-down assay, SUMOylated GST-XRCC1 was incubated with recombinant His-tagged POLB protein, followed by IP with anti-POLB antibody and IB with anti-GST and anti-POLB antibody (H). These blots has been cropped. To further clarify efficient recruitment of POLB by SUMOylated XRCC1 to promote BER activity, recombinant GST-tagged XRCC1 protein was used in in vitro SUMOylation assay and was further incubated with recombinant His-tagged POLB protein, followed by being analyzed by an immunoblotting assay and the in vitro BER assay (Fig. 6D). Using pull-down experiments, we demonstrated that SUMOylated XRCC1 pulled down more His-tagged POLB protein and had a higher BER activity compared to unmodified XRCC1 (Fig. 6E and F). It is notable that the lower BER activity in unmodified XRCC1 compared to SUMOylated XRCC1 was restored by supplemented with recombinant His-tagged POLB protein. Consistently, we also found that POLB preferentially bound to SUMOylated-XRCC1 as compared with unmodified XRCC1 based on a far-western blotting and an in vitro pull-down assay (Fig. 6G and H). These results suggested that the SUMOylated form of XRCC1 may have greater binding affinity for POLB. Taken together, these results suggest that SUMOylation regulates XRCC1 function in BER through more efficient interaction with POLB. Discussion PTM is of particular importance in the regulation of DDR and DNA repair through changes in protein localization, activity, or protein–protein interactions (9,40). As XRCC1 is an essential scaffold protein without enzymatic activity in BER, PTM is expected to be crucial for regulating the efficiency of XRCC1 to recruit other repair proteins. In this study, we have found that MMS increased SUMOylated XRCC1 endogenously in 293T cells and that K176 is the major SUMO acceptor on XRCC1. We demonstrated that MMS-induced PARylation promotes SUMOylation of XRCC1 and then SUMOylated XRCC1 increases the recruitment of the BER protein POLB to facilitate BER and cell survival after DNA damage, an effect that was partial but significant. In the DDR, PARylation usually has an early role, and PARylation of a protein can promote the formation of a molecular scaffold that facilitates the recruitment of other functionally linked proteins that are required to complete specific functions more efficiently (29). In studying the regulatory mechanism of XRCC1 SUMOylation in cells, we found that MMS-mediated SUMOylation of XRCC1 relies on the activation of PARP1. Our data showed that either suppression of PARP1 activity or loss of PAR binding through an XRCC1 mutation decreased MMS-induced XRCC1 SUMOylation. We also demonstrated that SUMOylation of XRCC1 was increased by pre-incubation with biotin-PAR or PARylated PARP1 in a cell-free SUMOylation system. Moreover, we found that MMS-activated PARylation promoted XRCC1 SUMOylation by mediating the interaction between XRCC1 and its SUMO E3 ligase TOPORS, which was decreased in the presence of the PARP inhibitor. Cumulatively, our data suggest that PAR functions as a primary protein-binding matrix that facilitates the recruitment of TOPORS to XRCC1 and then promotes SUMOylation of XRCC1, which serves as a secondary scaffold to facilitate the recruitment of repair protein POLB to damaged DNA. Correspondingly, cooperation among different types of PTM has become the subject of intensive research. The interplay between PARylation and SUMOylation in response to genotoxic stress has been observed (41). For examples, genotoxic stress induced PARylation is required for the recruitment of SUMO E3 ligase PIASy and then triggered PAR-dependent IKKgamma SUMOylation (32). Here, our finding reports an important interaction between PARylation and SUMOylation which ensures XRCC1 to facilitate DNA repair. Studies of the complicated interplays between PTMs by ubiquitin, SUMO, and PAR in the DDR and DNA repair are of critical importance. Many cellular functions of PARylation are exerted through dynamic interactions of proteins that bind PAR via several PAR-binding modules (28,42). It has been shown that XRCC1 binds to PAR through a phosphate-binding pocket in the XRCC1 BRCT1 domain (39,43). Here, we found that PAR binds TOPORS specifically through its C terminus, containing an atypical PAR-binding RS domain (28,42). Our data showed that PAR mediates the recruitment of TOPORS to XRCC1 complex and thus promotes SUMOylation of XRCC1 to protect cells from MMS-induced DNA damage. Because PARP inhibitors are recently and increasingly used in targeted therapies for cancer, exploring the roles of both PAR-modified and PAR-binding proteins in the regulation of the DNA repair either directly or indirectly through regulating other PTMs (e.g. SUMOylation) will provide valuable insights into therapeutic strategies for cancer. XRCC1 plays an important role in the recruitment of BER enzymes to sites of DNA damage by interacting with POLB via its NTD (residues 1–183) (6) and by interacting with LIG3 via its BRCT II domain (residues 538–633) (8). Consistent with our finding that the major SUMO site is K176, which is located within the XRCC1 NTD, we observed a distinct preference for POLB to bind with SUMOylated-XRCC1 and a decrease in BER activity in the SUMOylation-defective mutant XRCC1 K176R. Our results are consistent with other reports showing that the NTD of XRCC1 is critical for the interaction with and recruitment of POLB to sites of damaged DNA (44) and that mutant forms of XRCC1 that affect the XRCC1 NTD and thus POLB interactions are deficient in the repair of MMS-induced DNA damage and exhibit a reduced rate of BER and survival (45). Compared with these earlier studies, our present study has provided deeper insights into SUMO-mediated regulation of XRCC1 function in BER through interactions with POLB and suggests an alternative mechanism by which the recruitment of repair proteins and DNA repair efficiency can be modulated. Materials and Methods Cell culture and transfection Human 293T cells were cultured in DME medium supplemented with 10% fetal bovine serum (FBS). Chinese hamster ovary EM9 (XRCC1-null) cells were cultured in alpha minimum essential medium supplemented with 10% FBS. All growth media were purchased from Sigma–Aldrich. The cells used in the study were authenticated by Genelabs (Taipei, Taiwan) to ensure the absence of contamination. Plasmid DNA constructs and small interfering RNAs (siRNAs), that were validated and published, were transiently transfected into 293T cells with Lipofectamine 2000 (Invitrogen). To restore XRCC1 in EM9 cells, FLAG-tagged WT or mutant XRCC1 was introduced into EM9 cells, and then stable clones were selected and maintained in the presence of 0.2 mg/ml G418. Preparation of cell extracts, IB, and IP To further characterize XRCC1 SUMOylation, cells were boiled in SDS-lysis buffer (0.5% SDS; 1 mM EDTA; 50 mM Tris–HCl, pH 8) and briefly sonicated, and the cell lysate was used for IP and IB. To determine protein interactions, cells were lysed in TEGN lysis buffer (20 mM Tris–HCl, pH 8; 1 mM EDTA; 0.5% [w/v] Nonidet P-40; 150 mM NaCl; 10% [w/v] glycerol; complete protease inhibitor cocktail from Roche; and phosphatase inhibitor Cocktails I and II from Sigma–Aldrich) and sonicated briefly. The resulting cell lysate was used for IP and IB. Bands were visualized by chemiluminescence imaging using ImageQuant LAS 4000 mini (GE Healthcare Life Sciences). In vitro SUMOylation and mass spectrometry analysis Purified full-length GST-XRCC1-His or segments of the protein (2 μg) were subjected to SUMOylation reactions as described (46) and analyzed with IB. The expression and purification of recombinant SUMOylated-XRCC1 in an E. coli system harboring a SUMO system plasmid was as described (47). The XRCC1 SUMOylation site(s) of recombinant SUMOylated-XRCC1 was analyzed by mass spectrometry as described (47). Survival, colony formation assay, and MTT assay Sensitivity to DNA-damaging agents was determined with a standard colony formation assay and MTT assay (13). Briefly, EM9-derived XRCC1 WT or K176R stable cells were seeded in 6–8–10 days, and then stained with crystal violet. Each assay was performed in triplicate. Survival rate was presented relative to control treatment (100%). For survival analysis using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, cells were seeded in a 24-well plate. The following day, cells were pre-treated with a range of 4-AN concentrations for 30 min and were co-treated with MMS for an additional 15 min. Treated cells are left to recover in growth media and incubated at 37°C for 3–4 days, and then the MTT assay was performed. Alkaline single-cell agarose gel electrophoresis (comet assay) The cells were seeded overnight, treated with MMS (0.1 mg/ml) for 15 min to induce DNA damage, and then incubated in fresh medium for 1 h to allow DNA repair. Cells were collected, and the alkaline comet assay was performed with the Comet Assay kit (Trevigen). The cells were assessed with fluorescence microscopy and analyzed using CometScore (TriTek Corp.). Typically, ≥100 cells per sample were used to calculate the tail moment. Quantitative real-time analysis of BER activity Cell extracts from EM9-derived XRCC1 WT or K176R stable cells were prepared for the in vitro BER assay as described (48,49). Briefly, in vitro BER assay was performed in 20-μl reactions containing 0.1 pmol synthetic nicked duplex DNA substrate (template B) and intact duplex DNA substrate (template C), and the reactions were incubated for 30 min at 37°C with crude cell extract. Then, 0.5% of the reaction product was subjected to real-time TaqMan PCR assays to quantify. Relative BER activity was normalized to the control (template C). For pull-down and BER assays, GST beads bound SUMOylated-XRCC1 reaction product (as mentioned earlier) was incubated with His-tagged POLB recombinant protein. The pulled down XRCC1 complex was subsequently analyzed by IB with anti-POLB and 1/10 volume IP’d product was subjected to BER assay supplement with T4 DNA ligase (NEB). In vitro binding assay and far-western blotting For in vitro binding assays, the SUMOylated-XRCC1 reaction product (as mentioned earlier) was subsequently incubated with His-tagged POLB recombinant protein. Subsequently, the immunoprecipitation assays with anti-POLB antibody or GST pull down assay were performed. The immunoprecipitated proteins were analyzed by IB with anti-POLB and anti-His or anti-GST antibodies. For far-western blotting, the SUMOylated-XRCC1 reaction product was separated by SDS–PAGE and transferred to duplicate membranes. The duplicate membranes were probed with His-tagged POLB recombinant protein and then separately incubated with anti-POLB or anti-GST antibodies in the standard steps in IB. In vitro PARylation PARylation reaction (40 μl) contains 50 mM Tris–HEPES at pH 8.0, 0.15 mM KCl, 10 mM MgCl2, 0.5 mM β-NAD (Sigma–Aldrich), 1× activated DNA (Trevigen), and PARP1 enzyme (Trevigen) as described (50). The reaction lacks β-NAD was used as the PARylation negatively control. PARylation was monitored by IB using anti-PAR antibody (Trevigen). 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( 2010) A new approach utilizing real-time qPCR to detect in vitro base excision repair. DNA Repair (Amst) , 9, 898– 906. Google Scholar CrossRef Search ADS PubMed  50 Legrand A.J., Choul-Li S., Spriet C., Idziorek T., Vicogne D., Drobecq H., Dantzer F., Villeret V., Aumercier M. ( 2013) The level of Ets-1 protein is regulated by poly(ADP-ribose) polymerase-1 (PARP-1) in cancer cells to prevent DNA damage. PLoS One , 8, e55883. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Molecular Genetics Oxford University Press

SUMOylation of XRCC1 activated by poly (ADP-ribosyl)ation regulates DNA repair

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© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0964-6906
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1460-2083
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10.1093/hmg/ddy135
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Abstract

Abstract XRCC1 is an essential scaffold protein for base excision repair (BER) and helps to maintain genomic stability. XRCC1 has been indicated as a substrate for small ubiquitin-like modifier modification (SUMOylation); however, how XRCC1 SUMOylation is regulated in cells and how SUMOylated XRCC1 regulates BER activity are not well understood. Here, we show that SUMOylation of XRCC1 is regulated in cells under methyl-methanesulfonate (MMS) treatment and facilitates BER. Poly(ADP-ribose) polymerase 1 (PARP1) is activated by MMS immediately and synthesizes poly(ADP-ribose) (PAR), which in turn promotes recruitment of SUMO E3 TOPORS to XRCC1 and facilitates XRCC1 SUMOylation. A SUMOylation-defective mutant of XRCC1 had lower binding activity for DNA polymerase beta (POLB) and was linked to a lower capacity for repair of MMS-induced DNA damages. Our study therefore identified a pathway in which DNA damage-induced poly(ADP-ribosyl)ation (PARylation) promotes SUMOylation of XRCC1, which leads to more efficient recruitment of POLB to complete BER. Introduction Base excision repair (BER) fixes DNA base lesions and single-strand breaks (SSBs), which are caused by ubiquitous endogenous and exogenous agents, including reactive oxygen species, alkylating agents, and ionizing irradiation. Impaired BER results in both a decreased cellular capacity to counter genotoxic stress and increased genomic instability. BER is initiated by excision of the damaged base by DNA glycosylases and subsequently leads to a SSB, which is then filled by DNA polymerase β (POLB) and DNA ligase III (LIG3) (1). When SSBs are formed, the DNA-damage response (DDR) is activated to promote efficient BER via phosphorylation of the BER proteins by ATM/ATR-associated signals (2) and poly(ADP-ribosyl)ation (PARylation) of multiple BER proteins by poly(ADP-ribose) polymerases (PARPs) (3). XRCC1 plays a central role in BER, and its loss in cells leads to increased chromosomal instability and impaired DNA repair (4). Interestingly, XRCC1 itself has no enzymatic activity, but the scaffold function of XRCC1 is essential for BER (4,5). XRCC1 specifically interacts with other BER components through different domains: the N-terminal domain (NTD) interacts with POLB (6), the BRCA1 C-terminal (BRCT) I domain interacts with PARP (7), and the BRCT II domain interacts with LIG3 (8). Therefore, we wished to identify the regulatory mechanisms that control XRCC1 interactions with other BER components. Post-translational modification (PTM) of proteins by PARylation, phosphorylation, ubiquitination, and small ubiquitin-like modifier modification (SUMOylation) is a well-recognized mechanism that dynamically regulates the cellular functions of proteins, including not only the cell cycle and apoptosis but also the DDR and DNA repair (9). In particular, the formation of the repair complex is influenced by DNA damage–induced PTMs, which increases the specificity and efficiency of BER (10). Several PTMs were recently identified as mechanisms involved in the regulation of BER proteins in response to DNA damage (10). PTM of XRCC1 for DDR and DNA repair occurs via phosphorylation, ubiquitination, and PARylation. For example, phosphorylation at multiple sites within XRCC1 by protein kinases CK2 and Chk2 is required to maintain its stability and promote efficient DNA repair, respectively (11–13). XRCC1 is also ubiquitinated by the E3 ligase CHIP during the steady state (14) and by Iduna when DNA damage occurs (15); PARylation of XRCC1 prevents DNA damage-induced ubiquitylation (16). These observations suggest the importance of multiple PTMs of XRCC1 during BER. Protein SUMOylation has recently been suggested as a PTM that is critical for DDR and DNA repair (9). The SUMO proteins, SUMO-1, -2, and -3, share structural similarities with ubiquitin and form isopeptide linkages with specific lysine residues within the consensus motif Ψ-K-x-D/E (Ψ is a hydrophobic amino acid and x is any amino acid residue) in their substrates through an enzymatic cascade involving the sequential action of E1 activator, E2 conjugase, and E3 ligase (17). SUMO proteins are proposed to regulate the function of DNA repair proteins, including thymine-DNA glycosylase (18), BRCA1 (19), BLM (20), RPA70 (21), and MDC1 (22). Because protein SUMOylation is important in DNA repair and because PTM of XRCC1 is critical for its function in BER, we hypothesized that XRCC1 may increase SUMOylation upon DNA damage and that this PTM may be involved in the regulation of XRCC1 function in BER via the impact on repair complex formation and DNA repair efficiency. In this study, we found that the DNA alkylating-agent methyl methanesulfonate (MMS) induces PARP1 activation and PAR synthesis, and subsequently recruits SUMO E3 TOPORS to the XRCC1 complex. Furthermore, the interaction between TOPORS and XRCC1 facilitates XRCC1 SUMOylation, which leads to more-efficient BER via its ability to efficiently bind with POLB and thus contributes to cell survival after MMS-induced damage. Results DNA alkylating-agent MMS increases XRCC1 SUMOylation Although XRCC1 was reported to be SUMOylated both in vitro and in vivo (23–26), it is unclear how XRCC1 SUMOylation is regulated in cells. To understand this, we explored whether endogenous XRCC1 SUMOylation can be regulated in cells treated with MMS. Immunoblotting (IB) analysis of immunoprecipitated endogenous XRCC1 from 293T cells revealed that one major and one minor slower-migrating bands of XRCC1 were increased by MMS treatment (Fig. 1A, right, asterisk and double asterisks, respectively). The bands were also detected by anti-SUMO1 and anti-SUMO2/3 antibodies (Fig. 1B and C). The endogenous XRCC1 SUMOylation increased by MMS was also demonstrated by IP-IB analysis in breast cancer cell line MCF-7 (Supplementary Material, Fig. S1). Similar results were obtained with overexpression of FLAG-XRCC1 and EGFP-SUMO1 (Fig. 1D) or EGFP-SUMO2 (Fig. 1E). We further demonstrated that these two bands were SUMOylated XRCC1 because UBC9 knockdown notably reduced the slowly migrating XRCC1 bands (Fig. 1F). These results suggested that MMS treatment upregulated XRCC1 SUMOylation. Figure 1. View largeDownload slide  SUMOylation of XRCC1 was increased in response to alkylating DNA damage. (A–C) SUMOylated XRCC1 was increased by MMS treatment. 293T cells were treated with MMS (0.3 mg/ml for 1 h), and cell lysates were prepared and immunoprecipitated with anti-XRCC1 and immunoblotted with anti-XRCC1 (A), anti-SUMO1 (B), or anti-SUMO2/3 (C). Left panel in (A), immunoblots show 2.5% input of each lysate subjected to the immunoprecipitation (IP) assay with the indicated antibodies. Normal IgG was used as a negative control for IP assays. The ratio of SUMO-modified/unmodified XRCC1 band was indicated under the blots. (D and E) Immunoblots show MMS increased SUMOylation of XRCC1 in 293T cells that overexpressed FLAG-XRCC1 and EGFP-SUMO1 (D) or EGFP-SUMO2 (E). (F) Immunoblots show that SUMOylation of XRCC1 was reduced by siRNA-mediated knockdown of UBC9 in 293T cells with (+) and without (C) MMS treatment. (G) An in vitro SUMOylation assay was performed with the recombinant protein N-terminal GST-tagged and C-terminal His-tagged XRCC1, His-tagged SUMO1, and SUMOylation components. The in vitro SUMOylation reaction was performed and analyzed by immunoblotting with anti-His antibody. (H) Immunoblots show that the consensus SUMO motif VKEE in XRCC1 is the major acceptor for SUMOylation. SUMOylation of XRCC1 was abolished in 293T cells co-transfected with FLAG-XRCC1 mutant K176R or E178A along with EGFP-SUMO1. The SUMOylated XRCC1 bands were analyzed by immunoblotting (IB) with anti-FLAG and anti-GFP antibodies. In immunoblots here and throughout the figures, an arrowhead indicates unmodified XRCC1, an asterisk (*) indicates the major SUMO-modified XRCC1 band, double asterisks (**) indicate the minor SUMO-modified XRCC1 band, an arrow indicates a non-specific band, numbers to the side of the blots indicate molecular weight in kilodalton (kDa)s, anti-Tubulin or anti-Lamin B are used as loading control, MMS-induced DNA damage is indicated with anti-Chk2 (phospho-T68) or anti-p53 (phospho-S15), and C or Control indicates that dimethyl sulfoxide (DMSO) was used as the vehicle control. Figure 1. View largeDownload slide  SUMOylation of XRCC1 was increased in response to alkylating DNA damage. (A–C) SUMOylated XRCC1 was increased by MMS treatment. 293T cells were treated with MMS (0.3 mg/ml for 1 h), and cell lysates were prepared and immunoprecipitated with anti-XRCC1 and immunoblotted with anti-XRCC1 (A), anti-SUMO1 (B), or anti-SUMO2/3 (C). Left panel in (A), immunoblots show 2.5% input of each lysate subjected to the immunoprecipitation (IP) assay with the indicated antibodies. Normal IgG was used as a negative control for IP assays. The ratio of SUMO-modified/unmodified XRCC1 band was indicated under the blots. (D and E) Immunoblots show MMS increased SUMOylation of XRCC1 in 293T cells that overexpressed FLAG-XRCC1 and EGFP-SUMO1 (D) or EGFP-SUMO2 (E). (F) Immunoblots show that SUMOylation of XRCC1 was reduced by siRNA-mediated knockdown of UBC9 in 293T cells with (+) and without (C) MMS treatment. (G) An in vitro SUMOylation assay was performed with the recombinant protein N-terminal GST-tagged and C-terminal His-tagged XRCC1, His-tagged SUMO1, and SUMOylation components. The in vitro SUMOylation reaction was performed and analyzed by immunoblotting with anti-His antibody. (H) Immunoblots show that the consensus SUMO motif VKEE in XRCC1 is the major acceptor for SUMOylation. SUMOylation of XRCC1 was abolished in 293T cells co-transfected with FLAG-XRCC1 mutant K176R or E178A along with EGFP-SUMO1. The SUMOylated XRCC1 bands were analyzed by immunoblotting (IB) with anti-FLAG and anti-GFP antibodies. In immunoblots here and throughout the figures, an arrowhead indicates unmodified XRCC1, an asterisk (*) indicates the major SUMO-modified XRCC1 band, double asterisks (**) indicate the minor SUMO-modified XRCC1 band, an arrow indicates a non-specific band, numbers to the side of the blots indicate molecular weight in kilodalton (kDa)s, anti-Tubulin or anti-Lamin B are used as loading control, MMS-induced DNA damage is indicated with anti-Chk2 (phospho-T68) or anti-p53 (phospho-S15), and C or Control indicates that dimethyl sulfoxide (DMSO) was used as the vehicle control. We next delineated the XRCC1 SUMOylation site(s). We performed an in vitro SUMOylation assay with XRCC1 recombinant protein. The results also showed one major and one minor SUMOylated XRCC1 band (Fig. 1G and Supplementary Material, Fig. S2). The major SUMOylated XRCC1 band was further subjected to mass spectrometry analysis and revealed K176 as a SUMOylation site (Supplementary Material, Fig. S2C), consistent with a report showing that XRCC1 was SUMOylated by SUMO2 (25) and the XRCC1 K176R mutant was defective in SUMOylation based on an Escherichia coli SUMOylation system (26). We further confirmed K176 as the major SUMOylation site in cells with MMS treatment (Fig. 1H). This XRCC1 SUMOylation site, VKEEDE, is consistent with the SUMOylation consensus motif Ψ-K-x-D/E (27). Mutation of acidic residue E within this motif should reduce the XRCC1 SUMOylation. As expected, the E178A mutant decreased XRCC1 SUMOylation in cells (Fig. 1H). Note that the minor SUMOylated band of XRCC1 was not affected by the K176R or E178A mutation. MMS-induced SUMOylation of XRCC1 is regulated by PARylation To provide a more comprehensive view, we investigated whether the MMS-induced DDR signaling is involved in the regulation of XRCC1 SUMOylation. PARylation, an immediately-early reaction of DDR, is activated by PARPs in response to genotoxic stress, and after completing its effect, the degradation of poly-(ADP-ribose) (PAR) chain is regulated by the opposing reaction of PARG (28). If, indeed, XRCC1 SUMOylation is regulated by PARylation, it would be expected that the level of XRCC1 SUMOylation would decrease in cells with suppressed PARP activity or would increase with suppressed PARG activity. Thus, we manipulated the activity of PARP and PARG in 293T cells to provide evidence for the involvement of PARylation in XRCC1 SUMOylation. Indeed, SUMOylation of XRCC1 was decreased when PARP activity was inhibited by the PARP-specific inhibitor 4-amino-1,8-naphthalimide (4-AN) and increased in cells treated with PARG inhibitor tannic acid (Fig. 2A). Given that PARP1 is the most important member of PARP family for the synthesis of PAR chains (29), we demonstrated that siRNA knockdown of endogenous PARP1 reduced MMS-induced XRCC1 SUMOylation (Fig. 2B); whereas knockdown of PARG increased XRCC1 SUMOylation (Fig. 2C). Consistent to this expectation, the endogenous level of MMS-induced XRCC1 SUMOylation was decreased by PARP inhibitor 4-AN (Fig. 2D). Taken together, these results demonstrated that MMS-induced SUMOylation of XRCC1 is regulated by PARylation, which is mainly catalyzed by PARP1. Figure 2. View largeDownload slide  MMS-induced SUMOylation of XRCC1 is regulated by PARylation. (A) Immunoblots show that SUMOylation of XRCC1 is modulated by a PARylation-related inhibitor. 293T cells that co-expressed FLAG-XRCC1 and EGFP-SUMO1 were treated with MMS after pre-treatment with a specific PARP inhibitor (4-AN, 10μM) or PARG inhibitor (tannic acid, 100 μM) for 1 h. (B) Immunoblots show that SUMOylation of XRCC1 was decreased by knockdown of PARP1 with a specific siRNA in 293T cells co-transfected with FLAG-XRCC1 and EGFP-SUMO1. (C) MMS-induced SUMOylation of XRCC1 was increased by depletion of PARG. Prior to MMS treatment, 293T cells were transfected with PARG-specific siRNA along with FLAG-XRCC1 and EGFP-SUMO1. The lysates from transfected cells were analyzed by IB with anti-GFP and anti-FLAG antibodies. The efficiency of PARG knockdown was quantified with real-time PCR and normalized to the expression of GAPDH. Data represent the mean ± SD (Student’s t-test.) from two experiments performed in triplicate. (D) Immunoblots of immunoprecipitated endogenous XRCC1 show that MMS-increased SUMOylated-XRCC1 was reduced by PARP inhibitor 4-AN. 293T cells were pretreatment with or without 4-AN (10 μM) for 1 h and then continued incubation following MMS for 1 h, and then cell lysates were subjected to IP with anti-XRCC1 antibody. These blots has been cropped and the full-length blots of IPs (D, right panel) were included in Supplementary Material. Figure 2. View largeDownload slide  MMS-induced SUMOylation of XRCC1 is regulated by PARylation. (A) Immunoblots show that SUMOylation of XRCC1 is modulated by a PARylation-related inhibitor. 293T cells that co-expressed FLAG-XRCC1 and EGFP-SUMO1 were treated with MMS after pre-treatment with a specific PARP inhibitor (4-AN, 10μM) or PARG inhibitor (tannic acid, 100 μM) for 1 h. (B) Immunoblots show that SUMOylation of XRCC1 was decreased by knockdown of PARP1 with a specific siRNA in 293T cells co-transfected with FLAG-XRCC1 and EGFP-SUMO1. (C) MMS-induced SUMOylation of XRCC1 was increased by depletion of PARG. Prior to MMS treatment, 293T cells were transfected with PARG-specific siRNA along with FLAG-XRCC1 and EGFP-SUMO1. The lysates from transfected cells were analyzed by IB with anti-GFP and anti-FLAG antibodies. The efficiency of PARG knockdown was quantified with real-time PCR and normalized to the expression of GAPDH. Data represent the mean ± SD (Student’s t-test.) from two experiments performed in triplicate. (D) Immunoblots of immunoprecipitated endogenous XRCC1 show that MMS-increased SUMOylated-XRCC1 was reduced by PARP inhibitor 4-AN. 293T cells were pretreatment with or without 4-AN (10 μM) for 1 h and then continued incubation following MMS for 1 h, and then cell lysates were subjected to IP with anti-XRCC1 antibody. These blots has been cropped and the full-length blots of IPs (D, right panel) were included in Supplementary Material. PAR-mediated SUMO E3 ligase TOPORS recruitment to XRCC1 promotes SUMOylation To explore in-depth mechanisms of the role of PARylation in XRCC1 SUMOylation, we investigated how PARylation facilitates XRCC1 SUMOylation. Upon MMS treatment, PARP-mediated PARylation is the DDR that helps to recruit proteins to sites of DNA damage to promote efficient BER (30). PAR can serve as a molecular matrix to promote protein–protein interactions (28,31). Protein PARylation has been suggested to regulate SUMOylation of IKKgamma by recruitment of SUMO E3 ligase PIASy in cells under genotoxic stress (32). Given our finding above that XRCC1 is a SUMOylated target, the next question would be which SUMO E3 ligases regulate SUMOylation of XRCC1 and whether PAR is involved in the interaction between XRCC1 and E3 ligase. The SUMO E3 ligases, including TOPORS (33,34), PIAS1 (35), CBX4 (also referred to as Pc2) (36), and MMS21 (37), have been reported to be involved in responses to DNA damage. We examined whether the protein level of these E3 ligases is notably altered after MMS treatment. Importantly, the endogenous level of TOPORS, but not of PIAS1, CBX4, and MMS21, was gradually increased by MMS treatment and returned to baseline levels after 2 h (Fig. 3A). We then performed knockdown experiments and found TOPORS to be the most important SUMO E3 ligase for XRCC1 (Fig. 3B). Additionally, we confirmed that the increase in TOPORS level upon MMS treatment was not due to regulation at the transcriptional level, as assessed by real-time qPCR (Supplementary Material, Fig. S3). These results indicated that SUMOylation of XRCC1 was correlated with the protein level of TOPORS and led us to suggest TOPORS is a SUMO E3 ligase for XRCC1. Figure 3. View largeDownload slide  PAR-mediated SUMO E3 ligase TOPORS recruitment to XRCC1 to promote SUMOylation. (A) Immunoblots show that the level of the SUMO E3 ligase TOPORS was obviously altered by MMS treatment in this time-course experiment, but not the level of CBX4, PIAS1, or MMS21. (B) Immunoblots show that the level of SUMOylated XRCC1 was decreased by knockdown of TOPORS with a specific siRNA, but not by knockdown of CBX4 or MMS21. (C) Endogenous XRCC1 and TOPORS were co-immunoprecipitated by anti-PAR antibody after MMS treatment. 293T cells were treated with MMS for indicated time periods in the absence or in the presence of 4-AN (pretreatment with 4-AN, 10 μM for 1 h), and then cell lysates were subjected to IP with anti-PAR antibody. Proteins in the immunoprecipitated complex were analyzed by IB with anti-PAR, anti-XRCC1, and anti-TOPORS antibodies. (D) Immunoblots show that the association of TOPORS with XRCC1 increased after MMS treatment in 293T cells transfected with FLAG-XRCC1 and Myc-TOPORS. (E) Immunoblots show that both PAR and TOPORS had increased association with XRCC1 upon treatment with MMS in 293T cells transfected with FLAG-XRCC1 and Myc-TOPORS, and then cell lysates were subjected to IP with anti-FLAG antibody. The association between TOPORS and XRCC1 was reduced by PARP inhibitor 4-AN. The blots were cropped and the full-length blots of IPs (E) were included in Supplementary Material. Figure 3. View largeDownload slide  PAR-mediated SUMO E3 ligase TOPORS recruitment to XRCC1 to promote SUMOylation. (A) Immunoblots show that the level of the SUMO E3 ligase TOPORS was obviously altered by MMS treatment in this time-course experiment, but not the level of CBX4, PIAS1, or MMS21. (B) Immunoblots show that the level of SUMOylated XRCC1 was decreased by knockdown of TOPORS with a specific siRNA, but not by knockdown of CBX4 or MMS21. (C) Endogenous XRCC1 and TOPORS were co-immunoprecipitated by anti-PAR antibody after MMS treatment. 293T cells were treated with MMS for indicated time periods in the absence or in the presence of 4-AN (pretreatment with 4-AN, 10 μM for 1 h), and then cell lysates were subjected to IP with anti-PAR antibody. Proteins in the immunoprecipitated complex were analyzed by IB with anti-PAR, anti-XRCC1, and anti-TOPORS antibodies. (D) Immunoblots show that the association of TOPORS with XRCC1 increased after MMS treatment in 293T cells transfected with FLAG-XRCC1 and Myc-TOPORS. (E) Immunoblots show that both PAR and TOPORS had increased association with XRCC1 upon treatment with MMS in 293T cells transfected with FLAG-XRCC1 and Myc-TOPORS, and then cell lysates were subjected to IP with anti-FLAG antibody. The association between TOPORS and XRCC1 was reduced by PARP inhibitor 4-AN. The blots were cropped and the full-length blots of IPs (E) were included in Supplementary Material. Based on the data mentioned earlier, we hypothesized that PAR is involved in the MMS-induced interaction between the SUMO E3 ligase TOPORS and XRCC1. To examine this, we assessed the binding of PAR with XRCC1 and TOPORS in 293T cells after MMS treatment. IB analysis of immunoprecipitated endogenous PAR revealed that XRCC1 and TOPORS were both present in the PAR complex, and their levels in the complex increased upon MMS treatment (Fig. 3C). These results also demonstrated in breast cancer cell lines MCF-7 and MDA-MB-453 cells (Supplementary Material, Fig. S4A and B). We further demonstrated that MMS treatment also increased the interaction between Myc-TOPORS and FLAG-XRCC1 as the result of a co-immunoprecipitation (co-IP) assay with anti-FLAG (Fig. 3D). Consistently, the recruitment of TOPORS to XRCC1 via PAR induced by MMS treatment was suppressed by the PARP inhibitor 4-AN and, as a result, the SUMOylation of XRCC1 was decreased by 4-AN in 293T cells that co-expressed FLAG-XRCC1 and Myc-TOPORS (Fig. 3E). These results led us to suggest that MMS-induced XRCC1 SUMOylation is regulated by PAR through the recruitment of SUMO E3 ligase TOPORS to XRCC1 to promote SUMOylation. PAR-binding of XRCC1 is crucial for MMS-induced XRCC1 SUMOylation We showed above that PAR mediated the formation of XRCC1 and TOPORS complex. Thus, in the following experiments, we examined the PAR-binding ability of XRCC1 and TOPORS is important for the association of XRCC1 and TOPORS. The C-terminus of TOPORS is required for SUMOylation via interaction with SUMO1 and UBC9 (38) and is a potential PAR-binding segment containing an RS domain (SR-rich domain), i.e. a potential PAR-binding domain (28) (Supplementary Material, Fig. S5A). Thus, we performed specific experiments and demonstrated that the C-terminus of TOPORS is required for its binding ability to XRCC1 and PAR (Supplementary Material, Fig. S5B–E), and is also required for XRCC1 SUMOylation in vitro (Supplementary Material, Fig. S6). Furthermore, the co-IP results showed that the level of MMS-induced XRCC1 SUMOylation and the interaction of XRCC1 and TOPORS-C were suppressed by the PARP inhibitor (Fig. 4A). XRCC1 binds to PAR through its BRCT I domain (31), and the PAR binding-defective XRCC1 mutant RK (R335A/K369A) reduces DNA repair activity and cell survival after MMS-induced damage (39). Thus, to clarify the involvement of PAR in promoting XRCC1 SUMOylation, biotin-PAR or in vitro PARylated-PARP1 was pre-incubated with recombinant GST-tagged XRCC1 protein, followed by an in vitro SUMOylation assay with purified HA-tagged TOPORS-C and SUMOylation components (Fig. 4B, left). Immunoblots showed SUMOylation of XRCC1 was increased by pre-incubation with either biotin-PAR or PARylated-PARP1, but not with PARP1 alone in the absence of NAD+ (Fig. 4B, right). To further confirm whether the PAR-binding activity of XRCC1 is required for SUMOylation, we examined the SUMOylation of XRCC1 in 293T cells that expressed FLAG-tagged WT or RK XRCC1 with EGFP-SUMO1. The PAR binding-defective mutant of XRCC1 attenuated MMS-induced XRCC1 SUMOylation (Fig. 4C) and reduced its interaction with PAR and TOPORS-C (Fig. 4D). We further demonstrated that XRCC1 SUMOylation was promoted by overexpression of full-length TOPORS. In contrast, the level of XRCC1 SUMOylation and the binding of XRCC1 to TOPORS were reduced in XRCC1 PAR binding-defective mutant (Fig. 4E). Taken together, these data provide additional support for the involvement of PARylation in promoting SUMOylation of XRCC1 by facilitating the formation of the XRCC1/TOPORS complex upon MMS treatment. Figure 4. View largeDownload slide  PAR-binding of XRCC1 is crucial for MMS-induced XRCC1 SUMOylation. (A) Immunoblots show that the association between XRCC1 and TOPORS-C decreased upon treatment with PARP inhibitor 4-AN. 293T cells transfected with HA-TOPORS-C and FLAG-XRCC1 were pre-treated with 4-AN for 1 h before treatment with MMS, and then cell lysates were subjected to IP with anti-FLAG antibody. (B) SUMOylation of XRCC1 was increased by addition of biotin-PAR and by pre-PARylated PARP1 in a cell-free system. GST-tagged XRCC1-His protein bound to GST beads was incubated with biotin-PAR or the PARylation product of PARP1. After washing, an in vitro SUMOylation assay was performed in the presence of purified HA-tagged TOPORS-C protein. The reaction was analyzed by IB with anti-SUMO1 and anti-GST antibodies. (C) Immunoblots show that SUMOylation of RK XRCC1 was decreased in 293T cells that co-expressed EGFP-SUMO1 with FLAG-tagged WT or RK XRCC1 after MMS treatment for 1 h. (D) Immunoblots show that the ability of XRCC1 to bind to TOPORS-C was decreased in the RK XRCC1 PAR-binding mutant. 293T cells transfected with HA-TOPORS-C and FLAG-XRCC1 were treated with MMS for 30 min, and then cell lysates were subjected to IP with anti-FLAG antibody. (E) Immunoblots show that the SUMOylation of XRCC1 was increased by overexpression of full-length TOPORS and the ability of XRCC1 to bind to TOPORS was higher in WT than in the RK XRCC1 PAR-binding mutant. 293T cells transfected with Myc-TOPORS, EGFP-SUMO1, and FLAG-XRCC1 were treated with MMS for 1 h, and then cell lysates were subjected to IP with anti-FLAG antibody. These blots has been cropped. Figure 4. View largeDownload slide  PAR-binding of XRCC1 is crucial for MMS-induced XRCC1 SUMOylation. (A) Immunoblots show that the association between XRCC1 and TOPORS-C decreased upon treatment with PARP inhibitor 4-AN. 293T cells transfected with HA-TOPORS-C and FLAG-XRCC1 were pre-treated with 4-AN for 1 h before treatment with MMS, and then cell lysates were subjected to IP with anti-FLAG antibody. (B) SUMOylation of XRCC1 was increased by addition of biotin-PAR and by pre-PARylated PARP1 in a cell-free system. GST-tagged XRCC1-His protein bound to GST beads was incubated with biotin-PAR or the PARylation product of PARP1. After washing, an in vitro SUMOylation assay was performed in the presence of purified HA-tagged TOPORS-C protein. The reaction was analyzed by IB with anti-SUMO1 and anti-GST antibodies. (C) Immunoblots show that SUMOylation of RK XRCC1 was decreased in 293T cells that co-expressed EGFP-SUMO1 with FLAG-tagged WT or RK XRCC1 after MMS treatment for 1 h. (D) Immunoblots show that the ability of XRCC1 to bind to TOPORS-C was decreased in the RK XRCC1 PAR-binding mutant. 293T cells transfected with HA-TOPORS-C and FLAG-XRCC1 were treated with MMS for 30 min, and then cell lysates were subjected to IP with anti-FLAG antibody. (E) Immunoblots show that the SUMOylation of XRCC1 was increased by overexpression of full-length TOPORS and the ability of XRCC1 to bind to TOPORS was higher in WT than in the RK XRCC1 PAR-binding mutant. 293T cells transfected with Myc-TOPORS, EGFP-SUMO1, and FLAG-XRCC1 were treated with MMS for 1 h, and then cell lysates were subjected to IP with anti-FLAG antibody. These blots has been cropped. SUMOylation of XRCC1 promotes efficient DNA repair and cell survival after MMS treatment XRCC1 is required for DNA repair and survival from toxic DNA lesions induced by MMS (4). After the understanding of SUMOylation of XRCC1 upon MMS treatment, we investigated whether SUMOylation affects the function of XRCC1 in repairing MMS-induced DNA damages and cell survival. To this end, we first established CHO-derived XRCC1-deficient EM9 cells that stably expressed either FLAG-tagged XRCC1 WT or the K176R mutant (Fig. 5A) and then assessed the MMS-induced base damage repair using the comet assay. Compared with XRCC1 WT, XRCC1 K176R significantly reduced DNA repair, as evidenced by retained tail DNA (Fig. 5B and Supplementary Material, Fig. S7A) and consequently reduced clonogenic cell survival (Fig. 5C and Supplementary Material, Fig. S7B). Similar result was also demonstrated in transiently transfected MDA-MB-453 breast cancer cells (Supplementary Material, Fig. S8). Furthermore, we showed that the difference in survival between MMS-treated XRCC1 WT and K176R mutant stable cells was decreased if cells were pre-treated with PARP inhibitor 4-AN (Fig. 5D). These data demonstrated that SUMOylation of XRCC1 facilitates DNA repair and survival, and further supports for the suggestion that XRCC1 SUMOylation in BER is mediated by MMS-activated PARylation. Figure 5. View largeDownload slide  SUMOylation of XRCC1 enhances DNA repair and prevents cell death. (A) Immunoblots show the stable expression of XRCC1 WT or K176R in CHO-derived XRCC1-deficient EM9 cells. Cells expressing the empty vector were used as a control. (B) Quantification of the MMS-induced damaged DNA (comet tail) shows that EM9 cells harboring K176R XRCC1 retained more damaged DNA after recovery. Cells were treated with MMS (0.1 mg/ml) for 15 min, and then allowed to recover for 1 h. The data represent the % of DNA in comet tail from three independent experiments. For each treatment, 80∼100 cells were quantified. The results were analyzed by Prism. ***, P < 0.001 based on t-test. (C) Cells harboring K176R XRCC1 were more sensitive to MMS treatment. EM9-derived XRCC1 WT or K176R stable cells were treated with various doses of MMS for 15 min and then examined for colony-forming ability. Survival is expressed as a percentage of the untreated control for each clone (mean ± SD) from three independent experiments performed in triplicate. The generalized linear model (GLM) was used to test for a linear relationship (trend) of WT and K176R XRCC1 cells after serial doses of MMS treatment. ***, P < 0.05 was the limit of significance. (D). The difference in MMS-induced cell death between WT and K176R XRCC1 cells was reduced by PARP inhibitor 4-AN. WT or K176R XRCC1 cells were incubated with various doses of 4-AN for 30 min and then were treated with MMS (0.15 mg/ml for 15 min) or a vehicle control. Cellular viability was measured with the MTT assay and is expressed as a percentage of the untreated control for each clone. Data are presented relative to untreated cells and represent the mean ± SD for one experiment performed in triplicate. These blots has been cropped. Figure 5. View largeDownload slide  SUMOylation of XRCC1 enhances DNA repair and prevents cell death. (A) Immunoblots show the stable expression of XRCC1 WT or K176R in CHO-derived XRCC1-deficient EM9 cells. Cells expressing the empty vector were used as a control. (B) Quantification of the MMS-induced damaged DNA (comet tail) shows that EM9 cells harboring K176R XRCC1 retained more damaged DNA after recovery. Cells were treated with MMS (0.1 mg/ml) for 15 min, and then allowed to recover for 1 h. The data represent the % of DNA in comet tail from three independent experiments. For each treatment, 80∼100 cells were quantified. The results were analyzed by Prism. ***, P < 0.001 based on t-test. (C) Cells harboring K176R XRCC1 were more sensitive to MMS treatment. EM9-derived XRCC1 WT or K176R stable cells were treated with various doses of MMS for 15 min and then examined for colony-forming ability. Survival is expressed as a percentage of the untreated control for each clone (mean ± SD) from three independent experiments performed in triplicate. The generalized linear model (GLM) was used to test for a linear relationship (trend) of WT and K176R XRCC1 cells after serial doses of MMS treatment. ***, P < 0.05 was the limit of significance. (D). The difference in MMS-induced cell death between WT and K176R XRCC1 cells was reduced by PARP inhibitor 4-AN. WT or K176R XRCC1 cells were incubated with various doses of 4-AN for 30 min and then were treated with MMS (0.15 mg/ml for 15 min) or a vehicle control. Cellular viability was measured with the MTT assay and is expressed as a percentage of the untreated control for each clone. Data are presented relative to untreated cells and represent the mean ± SD for one experiment performed in triplicate. These blots has been cropped. SUMOylation of XRCC1 facilitates DNA repair mediated through efficient POLB recruitment To further understand the XRCC1 SUMOylation-mediated-DNA repair, we provided supporting evidence by directly assessing the repair activity of specific BER members using cell extracts from these cell lines using the in vitro BER/SSBR assay (Fig. 6A). Similar to the observations with the comet assay, BER activity was significantly decreased in stable cells expressing the K176R mutant (Fig. 6B). The results implied that SUMOylation regulates the function of XRCC1 in BER. XRCC1 acts as a scaffold to recruit BER enzymes, including POLB and LIG3, to the sites of DNA damage, which is important for efficient damage repair (7). We hypothesized that SUMOylation may regulate the interaction of XRCC1 with BER repair proteins. To address this possibility, we assessed the binding of repair proteins POLB and LIG3 to WT and K176R XRCC1. The results of co-immunoprecipitation experiments showed that the XRCC1 K176R mutant notably decreased its interaction with POLB, but not LIG3 (Fig. 6C). Similar result was also demonstrated in MDA-MB-453 breast cancer cells (Supplementary Material, Fig. S9). Figure 6. View largeDownload slide SUMOylation of XRCC1 promotes BER activity mediated through efficient POLB recruitment. (A) Schematic representation of the process utilizing real-time PCR to quantify in vitro BER activity. Cell extract was incubated with synthesized template B (which includes a single-nucleotide gap) for the in vitro BER assay. Intact template C in the reaction was used as the input control. The repaired template B was quantified with TaqMan-based real-time PCR. (B) The XRCC1 K176R mutant had lower BER activity. The cell extract from EM9-derived XRCC1 WT or K176R stable cells was incubated with template B and control template C. Relative BER activity was normalized to the control (template C). Data are presented relative to WT XRCC1 (100%) and represent the mean ± SD for two independent experiments performed in triplicate. ***, P < 0.001 based on Student’s t-test. (C) The association of POLB with XRCC1 decreased in the SUMOylation-defective K176R mutant. IB of the protein complex was carried out after IP of XRCC1 with anti-FLAG antibody from stable cells. The lysate was analyzed in parallel. (D–F) SUMOylated XRCC1 pulled down more POLB protein and had a higher BER activity compared to unmodified XRCC1. Schematic representation of the process utilizing in vitro SUMOylated-XRCC1 to pull-down His-tagged POLB protein and further analyze with IB and an in vitro BER assays (D). Immunoblots show that SUMOylated XRCC1 pulled down more POLB protein. Recombinant POLB protein was incubated with SUMOylated GST-XRCC1-His protein and then GST-pulled down. The GST-pull down products P1 and P2 were analyzed by IB with anti-POLB antibody (E). The difference of BER activity in these two repair complex can be restored by supplement with extra recombinant POLB protein. The GST-pull down products P1 and P2 were subjected to BER assay supplement with recombinant POLB protein (F). (G and H). POLB preferentially binds to SUMOylated-XRCC1, as determined by far-western blotting (G) and immuno-pull-down (H) assays. For the far-western blotting, in vitro SUMOylated GST-XRCC1 was separated by SDS–PAGE, immobilized on a nitrocellulose membrane, and incubated with recombinant His-tagged POLB protein as bait, followed by IB with anti-POLB antibody (G, right). The input level of GST-XRCC1 was analyzed with anti-GST antibody (G, left). The ratio of modified/unmodified bands indicates the binding of POLB to SUMO-modified versus SUMO-unmodified XRCC1. For the pull-down assay, SUMOylated GST-XRCC1 was incubated with recombinant His-tagged POLB protein, followed by IP with anti-POLB antibody and IB with anti-GST and anti-POLB antibody (H). These blots has been cropped. Figure 6. View largeDownload slide SUMOylation of XRCC1 promotes BER activity mediated through efficient POLB recruitment. (A) Schematic representation of the process utilizing real-time PCR to quantify in vitro BER activity. Cell extract was incubated with synthesized template B (which includes a single-nucleotide gap) for the in vitro BER assay. Intact template C in the reaction was used as the input control. The repaired template B was quantified with TaqMan-based real-time PCR. (B) The XRCC1 K176R mutant had lower BER activity. The cell extract from EM9-derived XRCC1 WT or K176R stable cells was incubated with template B and control template C. Relative BER activity was normalized to the control (template C). Data are presented relative to WT XRCC1 (100%) and represent the mean ± SD for two independent experiments performed in triplicate. ***, P < 0.001 based on Student’s t-test. (C) The association of POLB with XRCC1 decreased in the SUMOylation-defective K176R mutant. IB of the protein complex was carried out after IP of XRCC1 with anti-FLAG antibody from stable cells. The lysate was analyzed in parallel. (D–F) SUMOylated XRCC1 pulled down more POLB protein and had a higher BER activity compared to unmodified XRCC1. Schematic representation of the process utilizing in vitro SUMOylated-XRCC1 to pull-down His-tagged POLB protein and further analyze with IB and an in vitro BER assays (D). Immunoblots show that SUMOylated XRCC1 pulled down more POLB protein. Recombinant POLB protein was incubated with SUMOylated GST-XRCC1-His protein and then GST-pulled down. The GST-pull down products P1 and P2 were analyzed by IB with anti-POLB antibody (E). The difference of BER activity in these two repair complex can be restored by supplement with extra recombinant POLB protein. The GST-pull down products P1 and P2 were subjected to BER assay supplement with recombinant POLB protein (F). (G and H). POLB preferentially binds to SUMOylated-XRCC1, as determined by far-western blotting (G) and immuno-pull-down (H) assays. For the far-western blotting, in vitro SUMOylated GST-XRCC1 was separated by SDS–PAGE, immobilized on a nitrocellulose membrane, and incubated with recombinant His-tagged POLB protein as bait, followed by IB with anti-POLB antibody (G, right). The input level of GST-XRCC1 was analyzed with anti-GST antibody (G, left). The ratio of modified/unmodified bands indicates the binding of POLB to SUMO-modified versus SUMO-unmodified XRCC1. For the pull-down assay, SUMOylated GST-XRCC1 was incubated with recombinant His-tagged POLB protein, followed by IP with anti-POLB antibody and IB with anti-GST and anti-POLB antibody (H). These blots has been cropped. To further clarify efficient recruitment of POLB by SUMOylated XRCC1 to promote BER activity, recombinant GST-tagged XRCC1 protein was used in in vitro SUMOylation assay and was further incubated with recombinant His-tagged POLB protein, followed by being analyzed by an immunoblotting assay and the in vitro BER assay (Fig. 6D). Using pull-down experiments, we demonstrated that SUMOylated XRCC1 pulled down more His-tagged POLB protein and had a higher BER activity compared to unmodified XRCC1 (Fig. 6E and F). It is notable that the lower BER activity in unmodified XRCC1 compared to SUMOylated XRCC1 was restored by supplemented with recombinant His-tagged POLB protein. Consistently, we also found that POLB preferentially bound to SUMOylated-XRCC1 as compared with unmodified XRCC1 based on a far-western blotting and an in vitro pull-down assay (Fig. 6G and H). These results suggested that the SUMOylated form of XRCC1 may have greater binding affinity for POLB. Taken together, these results suggest that SUMOylation regulates XRCC1 function in BER through more efficient interaction with POLB. Discussion PTM is of particular importance in the regulation of DDR and DNA repair through changes in protein localization, activity, or protein–protein interactions (9,40). As XRCC1 is an essential scaffold protein without enzymatic activity in BER, PTM is expected to be crucial for regulating the efficiency of XRCC1 to recruit other repair proteins. In this study, we have found that MMS increased SUMOylated XRCC1 endogenously in 293T cells and that K176 is the major SUMO acceptor on XRCC1. We demonstrated that MMS-induced PARylation promotes SUMOylation of XRCC1 and then SUMOylated XRCC1 increases the recruitment of the BER protein POLB to facilitate BER and cell survival after DNA damage, an effect that was partial but significant. In the DDR, PARylation usually has an early role, and PARylation of a protein can promote the formation of a molecular scaffold that facilitates the recruitment of other functionally linked proteins that are required to complete specific functions more efficiently (29). In studying the regulatory mechanism of XRCC1 SUMOylation in cells, we found that MMS-mediated SUMOylation of XRCC1 relies on the activation of PARP1. Our data showed that either suppression of PARP1 activity or loss of PAR binding through an XRCC1 mutation decreased MMS-induced XRCC1 SUMOylation. We also demonstrated that SUMOylation of XRCC1 was increased by pre-incubation with biotin-PAR or PARylated PARP1 in a cell-free SUMOylation system. Moreover, we found that MMS-activated PARylation promoted XRCC1 SUMOylation by mediating the interaction between XRCC1 and its SUMO E3 ligase TOPORS, which was decreased in the presence of the PARP inhibitor. Cumulatively, our data suggest that PAR functions as a primary protein-binding matrix that facilitates the recruitment of TOPORS to XRCC1 and then promotes SUMOylation of XRCC1, which serves as a secondary scaffold to facilitate the recruitment of repair protein POLB to damaged DNA. Correspondingly, cooperation among different types of PTM has become the subject of intensive research. The interplay between PARylation and SUMOylation in response to genotoxic stress has been observed (41). For examples, genotoxic stress induced PARylation is required for the recruitment of SUMO E3 ligase PIASy and then triggered PAR-dependent IKKgamma SUMOylation (32). Here, our finding reports an important interaction between PARylation and SUMOylation which ensures XRCC1 to facilitate DNA repair. Studies of the complicated interplays between PTMs by ubiquitin, SUMO, and PAR in the DDR and DNA repair are of critical importance. Many cellular functions of PARylation are exerted through dynamic interactions of proteins that bind PAR via several PAR-binding modules (28,42). It has been shown that XRCC1 binds to PAR through a phosphate-binding pocket in the XRCC1 BRCT1 domain (39,43). Here, we found that PAR binds TOPORS specifically through its C terminus, containing an atypical PAR-binding RS domain (28,42). Our data showed that PAR mediates the recruitment of TOPORS to XRCC1 complex and thus promotes SUMOylation of XRCC1 to protect cells from MMS-induced DNA damage. Because PARP inhibitors are recently and increasingly used in targeted therapies for cancer, exploring the roles of both PAR-modified and PAR-binding proteins in the regulation of the DNA repair either directly or indirectly through regulating other PTMs (e.g. SUMOylation) will provide valuable insights into therapeutic strategies for cancer. XRCC1 plays an important role in the recruitment of BER enzymes to sites of DNA damage by interacting with POLB via its NTD (residues 1–183) (6) and by interacting with LIG3 via its BRCT II domain (residues 538–633) (8). Consistent with our finding that the major SUMO site is K176, which is located within the XRCC1 NTD, we observed a distinct preference for POLB to bind with SUMOylated-XRCC1 and a decrease in BER activity in the SUMOylation-defective mutant XRCC1 K176R. Our results are consistent with other reports showing that the NTD of XRCC1 is critical for the interaction with and recruitment of POLB to sites of damaged DNA (44) and that mutant forms of XRCC1 that affect the XRCC1 NTD and thus POLB interactions are deficient in the repair of MMS-induced DNA damage and exhibit a reduced rate of BER and survival (45). Compared with these earlier studies, our present study has provided deeper insights into SUMO-mediated regulation of XRCC1 function in BER through interactions with POLB and suggests an alternative mechanism by which the recruitment of repair proteins and DNA repair efficiency can be modulated. Materials and Methods Cell culture and transfection Human 293T cells were cultured in DME medium supplemented with 10% fetal bovine serum (FBS). Chinese hamster ovary EM9 (XRCC1-null) cells were cultured in alpha minimum essential medium supplemented with 10% FBS. All growth media were purchased from Sigma–Aldrich. The cells used in the study were authenticated by Genelabs (Taipei, Taiwan) to ensure the absence of contamination. Plasmid DNA constructs and small interfering RNAs (siRNAs), that were validated and published, were transiently transfected into 293T cells with Lipofectamine 2000 (Invitrogen). To restore XRCC1 in EM9 cells, FLAG-tagged WT or mutant XRCC1 was introduced into EM9 cells, and then stable clones were selected and maintained in the presence of 0.2 mg/ml G418. Preparation of cell extracts, IB, and IP To further characterize XRCC1 SUMOylation, cells were boiled in SDS-lysis buffer (0.5% SDS; 1 mM EDTA; 50 mM Tris–HCl, pH 8) and briefly sonicated, and the cell lysate was used for IP and IB. To determine protein interactions, cells were lysed in TEGN lysis buffer (20 mM Tris–HCl, pH 8; 1 mM EDTA; 0.5% [w/v] Nonidet P-40; 150 mM NaCl; 10% [w/v] glycerol; complete protease inhibitor cocktail from Roche; and phosphatase inhibitor Cocktails I and II from Sigma–Aldrich) and sonicated briefly. The resulting cell lysate was used for IP and IB. Bands were visualized by chemiluminescence imaging using ImageQuant LAS 4000 mini (GE Healthcare Life Sciences). In vitro SUMOylation and mass spectrometry analysis Purified full-length GST-XRCC1-His or segments of the protein (2 μg) were subjected to SUMOylation reactions as described (46) and analyzed with IB. The expression and purification of recombinant SUMOylated-XRCC1 in an E. coli system harboring a SUMO system plasmid was as described (47). The XRCC1 SUMOylation site(s) of recombinant SUMOylated-XRCC1 was analyzed by mass spectrometry as described (47). Survival, colony formation assay, and MTT assay Sensitivity to DNA-damaging agents was determined with a standard colony formation assay and MTT assay (13). Briefly, EM9-derived XRCC1 WT or K176R stable cells were seeded in 6–8–10 days, and then stained with crystal violet. Each assay was performed in triplicate. Survival rate was presented relative to control treatment (100%). For survival analysis using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, cells were seeded in a 24-well plate. The following day, cells were pre-treated with a range of 4-AN concentrations for 30 min and were co-treated with MMS for an additional 15 min. Treated cells are left to recover in growth media and incubated at 37°C for 3–4 days, and then the MTT assay was performed. Alkaline single-cell agarose gel electrophoresis (comet assay) The cells were seeded overnight, treated with MMS (0.1 mg/ml) for 15 min to induce DNA damage, and then incubated in fresh medium for 1 h to allow DNA repair. Cells were collected, and the alkaline comet assay was performed with the Comet Assay kit (Trevigen). The cells were assessed with fluorescence microscopy and analyzed using CometScore (TriTek Corp.). Typically, ≥100 cells per sample were used to calculate the tail moment. Quantitative real-time analysis of BER activity Cell extracts from EM9-derived XRCC1 WT or K176R stable cells were prepared for the in vitro BER assay as described (48,49). Briefly, in vitro BER assay was performed in 20-μl reactions containing 0.1 pmol synthetic nicked duplex DNA substrate (template B) and intact duplex DNA substrate (template C), and the reactions were incubated for 30 min at 37°C with crude cell extract. Then, 0.5% of the reaction product was subjected to real-time TaqMan PCR assays to quantify. Relative BER activity was normalized to the control (template C). For pull-down and BER assays, GST beads bound SUMOylated-XRCC1 reaction product (as mentioned earlier) was incubated with His-tagged POLB recombinant protein. The pulled down XRCC1 complex was subsequently analyzed by IB with anti-POLB and 1/10 volume IP’d product was subjected to BER assay supplement with T4 DNA ligase (NEB). In vitro binding assay and far-western blotting For in vitro binding assays, the SUMOylated-XRCC1 reaction product (as mentioned earlier) was subsequently incubated with His-tagged POLB recombinant protein. Subsequently, the immunoprecipitation assays with anti-POLB antibody or GST pull down assay were performed. The immunoprecipitated proteins were analyzed by IB with anti-POLB and anti-His or anti-GST antibodies. For far-western blotting, the SUMOylated-XRCC1 reaction product was separated by SDS–PAGE and transferred to duplicate membranes. The duplicate membranes were probed with His-tagged POLB recombinant protein and then separately incubated with anti-POLB or anti-GST antibodies in the standard steps in IB. In vitro PARylation PARylation reaction (40 μl) contains 50 mM Tris–HEPES at pH 8.0, 0.15 mM KCl, 10 mM MgCl2, 0.5 mM β-NAD (Sigma–Aldrich), 1× activated DNA (Trevigen), and PARP1 enzyme (Trevigen) as described (50). The reaction lacks β-NAD was used as the PARylation negatively control. PARylation was monitored by IB using anti-PAR antibody (Trevigen). Statistical analysis Student’s t-tests were used to determine the significance of differences between samples as indicated in etalures. P < 0.001 was the limit of significance. The generalized linear model (GLM) was used to test for a linear relationship (trend) of WT and K176R XRCC1 cells after serial doses of MMS treatment. P < 0.05 was the limit of significance. Information about protein expression constructs, siRNAs, and additional materials and methods can be found in Supplementary Material. Supplementary Material Supplementary Material is available at HMG online. Acknowledgements We thank the Proteomics Core Facility of the Institute of Biomedical Sciences, Academia Sinica for identifying SUMO modification sites of XRCC1. Conflict of Interest statement. None declared. Funding This work was supported by the Academia Sinica of Taiwan (to C.-Y.S.). References 1 Robertson A.B., Klungland A., Rognes T., Leiros I. 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Human Molecular GeneticsOxford University Press

Published: Apr 16, 2018

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