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ADP‐ribosylation of histone variant H2AX promotes base excision repair

2020; Springer Nature; Volume: 40; Issue: 2 Linguagem: Inglês

10.15252/embj.2020104542

1460-2075

Qian Chen, Chunjing Bian, Xin Wang, Xiuhua Liu, Muzaffer Ahmad Kassab, Yonghao Yu, Xiaochun Yu,

Calcium signaling and nucleotide metabolism

Article2 December 2020free access Source DataTransparent process ADP-ribosylation of histone variant H2AX promotes base excision repair Qian Chen Corresponding Author Qian Chen [email protected] orcid.org/0000-0003-3718-3720 Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA Search for more papers by this author Chunjing Bian Chunjing Bian Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA Search for more papers by this author Xin Wang Xin Wang Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA Search for more papers by this author Xiuhua Liu Xiuhua Liu Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA Search for more papers by this author Muzaffer Ahmad Kassab Muzaffer Ahmad Kassab Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA Search for more papers by this author Yonghao Yu Yonghao Yu Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Xiaochun Yu Corresponding Author Xiaochun Yu [email protected] orcid.org/0000-0001-9840-2670 Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA Search for more papers by this author Qian Chen Corresponding Author Qian Chen [email protected] orcid.org/0000-0003-3718-3720 Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA Search for more papers by this author Chunjing Bian Chunjing Bian Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA Search for more papers by this author Xin Wang Xin Wang Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA Search for more papers by this author Xiuhua Liu Xiuhua Liu Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA Search for more papers by this author Muzaffer Ahmad Kassab Muzaffer Ahmad Kassab Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA Search for more papers by this author Yonghao Yu Yonghao Yu Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Xiaochun Yu Corresponding Author Xiaochun Yu [email protected] orcid.org/0000-0001-9840-2670 Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA Search for more papers by this author Author Information Qian Chen *,1, Chunjing Bian1,†, Xin Wang1, Xiuhua Liu1, Muzaffer Ahmad Kassab1, Yonghao Yu2 and Xiaochun Yu *,1,† 1Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA 2Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA †Present address: Cedar-Sinai Medical Center, Los Angeles, CA, USA †Present address: Westlake University, Hangzhou, Zhejiang, China *Corresponding author. Tel: +1 0571 86980080; E-mail: [email protected] *Corresponding author. Tel: +1 0571 86980080; E-mail: [email protected] The EMBO Journal (2021)40:e104542https://doi.org/10.15252/embj.2020104542 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Optimal DNA damage response is associated with ADP-ribosylation of histones. However, the underlying molecular mechanism of DNA damage-induced histone ADP-ribosylation remains elusive. Herein, using unbiased mass spectrometry, we identify that glutamate residue 141 (E141) of variant histone H2AX is ADP-ribosylated following oxidative DNA damage. In-depth studies performed with wild-type H2AX and the ADP-ribosylation-deficient E141A mutant suggest that H2AX ADP-ribosylation plays a critical role in base excision repair (BER). Mechanistically, ADP-ribosylation on E141 mediates the recruitment of Neil3 glycosylase to the sites of DNA damage for BER. Moreover, loss of this ADP-ribosylation enhances serine-139 phosphorylation of H2AX (γH2AX) upon oxidative DNA damage and erroneously causes the accumulation of DNA double-strand break (DSB) response factors. Taken together, these results reveal that H2AX ADP-ribosylation not only facilitates BER repair, but also suppresses the γH2AX-mediated DSB response. Synopsis The role of histone ADP-ribosylation after DNA damage has remained elusive. Here, ADP-ribosylation of histone variant H2AX on a specific residue upon oxidative damage is found to not only promote base excision repair (BER), but to also suppress γH2AX-associated DNA double-strand break (DSB) responses. Glutamate residue 141 (E141) of histone H2AX is ADP-ribosylated following oxidative DNA damage. ADP-ribosylation on E141 mediates recruitment of the BER glycosylase Neil3 to the sites of oxidative damage. H2AX E141A mutation abolishes ADP-ribosylation and enhances serine-139 phosphorylation to create γH2AX upon oxidative damage. Oxidative damage-induced γH2AX formation in H2AX-E141A mutants causes erroneous accumulation of DSB response factors. Introduction The integrity of genomic DNA is constantly challenged by endogenous and exogenous hazards generating numerous DNA lesions in human cells daily. These DNA lesions, if not repaired precisely and timely, can lead to genomic instability (Mills et al, 2003; Motoyama & Naka, 2004; Gorgoulis et al, 2005; Tubbs & Nussenzweig, 2017). To deal with the risks of DNA damage, cells use a complicated molecular network to sense, signal, and repair DNA lesions, aka DNA damage response (DDR) (Zhou & Elledge, 2000; Harper & Elledge, 2007; Jackson & Bartek, 2009; Lord & Ashworth, 2012). One of the major components of DDR involves chromatin remodeling at the DNA lesions (Bao & Shen, 2007; Wang et al, 2007; Lukas et al, 2011; Soria et al, 2012; Price & D'Andrea, 2013), which is primarily achieved with histone modifications (Iizuka & Smith, 2003; van Attikum & Gasser, 2005; Vidanes et al, 2005; van Attikum & Gasser, 2009; Messner & Hottiger, 2011; Xu et al, 2012). The most prominent histone modifications induced by DNA damage occur on H2AX, a variant of canonical histone H2A (Rogakou et al, 1998; Fernandez-Capetillo et al, 2004; Ikura et al, 2007; Xiao et al, 2009; Jiang et al, 2010; Yuan et al, 2010; Pan et al, 2011; Revet et al, 2011; Mattiroli et al, 2012; Sone et al, 2014; Ikura et al, 2015). In response to DNA damage, especially DNA double-strand breaks (DSBs), H2AX is rapidly phosphorylated at serine 139 (S139) by PI3-like kinases including ATM, ATR, and DNA-PK (Burma et al, 2001; Ward & Chen, 2001; Celeste et al, 2003; Stiff et al, 2004). The phosphorylated H2AX is also known as γH2AX, which is recognized by the downstream mediators such as MDC1 assists in the recruitment of repair machinery for DSB repair (Paull et al, 2000; Celeste et al, 2003; Stucki et al, 2005; Lou et al, 2006). In addition to phosphorylation, other posttranslational modifications (PTMs) on histones, such as ubiquitination (Pan et al, 2011; Mattiroli et al, 2012), SUMOylation (Corujo & Buschbeck, 2018), methylation (Sone et al, 2014), and acetylation (Jiang et al, 2010), also regulate chromatin status adjacent to DNA lesions and facilitate DNA damage repair. Among all damage signals, ADP-ribosylation is one of the earliest signals generated at DNA lesions, and histones due to their proximity with the DNA function as primary substrates of ADP-ribosylation in response to DNA damage (Ogata et al, 1980; Messner et al, 2010; Messner & Hottiger, 2011; Leidecker et al, 2016). Protein ADP-ribosylation is catalyzed by a group of poly(ADP-ribosyl)ation polymerases (aka PARPs) (Hassa et al, 2006). These enzymes utilize NAD+ as the ADP-ribose donor and transfer ADP-ribose moiety to protein substrates (Kim et al, 2005). In human cells, 17 PARPs have been identified (Liu & Yu, 2015), and many of them are known to mediate ADP-ribosylation in response to DNA damage (Kraus, 2015). In particular, PARP1 mediates the majority of ADP-ribosylation in response to DNA damage (Leung, 2014). Of note, not all the amino acid residues can accept PARP1-mediated ADP-ribosylation. Mass spectrometry analyses of the substrate proteins show that glutamate (E), aspartate (D), tyrosine (Y), and serine (S) residues function as primary targets of PARP1-mediated ADP-ribosylation (Blanke et al, 1994; Bock et al, 2015; Bonfiglio et al, 2017; Chapman et al, 2013; Kraus, 2015; Matic et al, 2012; Pedrioli et al, 2018; Zhang et al, 2013). Recent studies have shown that other residues including lysine (K) (Altmeyer et al, 2009; Messner et al, 2010), arginine (R) (Laing et al, 2011), and cysteine (C) (West et al, 1985) may also be ADP-ribosylated through alternate mechanisms. In addition to PARP1, other PARPs, including PARP 2, 3, 7, and 10, also catalyze ADP-ribosylation during DDR, and thus may have overlapping functions (Vyas et al, 2014; Wei & Yu, 2016; Liu et al, 2017). The general biological functions of ADP-ribosylation in DDR have been studied extensively. It has been shown that ADP-ribosylation brings huge amounts of negative charge to the chromatin adjacent to DNA lesions, which may facilitate chromatin relaxation (Poirier et al, 1982; Tulin & Spradling, 2003). Moreover, several ADP-ribosylation binding motifs have been identified, suggesting that ADP-ribosylation, like phosphorylation, acts as a signal to mediate the recruitment of DNA damage repair machinery to DNA lesions for the repair (Liu & Yu, 2015; Wei & Yu, 2016; Liu et al, 2017). However, like protein phosphorylation, different ADP-ribosylation sites may mediate different steps of DNA damage repair (Liu et al, 2017). Due to lack of analytic approaches, the biological functions of site-specific ADP-ribosylation in DNA damage repair are not well studied. Fortunately, over the past few years, advanced mass spectrometry technologies have revealed more and more ADP-ribosylation sites (Altmeyer et al, 2009; Messner et al, 2010; Laing et al, 2011; Zhang et al, 2013; Bock et al, 2015; Leidecker et al, 2016; Pedrioli et al, 2018). In this study, using unbiased high-resolution mass spectrometry and other molecular biology approaches, we characterized the ADP-ribosylation of E141 of H2AX and provided insights into its critical role in base excision repair (BER). Results H2AX is ADP-ribosylated in response to DNA damage Since H2AX plays a key role in DNA damage response and protein ADP-ribosylation is one of the earliest signals at DNA lesions, we asked whether H2AX could act as an acceptor of ADP-ribose in response to DNA damage. We treated 293T cells with various DNA damaging agents, including H2O2, MMS, MMC, cisplatin, and ionizing radiation (IR), and examined the status of H2AX in the chromatin fractions. Using immunoprecipitation (IP) and Western blotting assays, we found that H2AX was clearly ADP-ribosylated in response to H2O2 and MMS treatment (Fig 1A). However, simulations with MMC, cisplatin, or IR did not induce noticeable H2AX ADP-ribosylation, indicating ADP-ribosylation on H2AX is a specific event induced by oxidative DNA damage (Appendix Fig S1A and B). Previous studies have shown that DNA damage-induced ADP-ribosylation is associated with the intensity of oxidative damage (Martello et al, 2013; Bilan et al, 2017). Here, we found that the ADP-ribosylation on H2AX was also remarkably increased along with the increased level of oxidative stress (Appendix Fig S2). Figure 1. H2AX is ADP-ribosylated in response to DNA damage DNA damage induces H2AX ADP-ribosylation. 293T cells were treated with H2O2 (2 mM in PBS, 5 min), MMS (1 mM in medium, 30 min), or mock (PBS, 30 min). ADP-ribosylated proteins were IPed with anti-ADP-ribose (anti-ADPR) antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody. H2AX is ADP-ribosylated at glutamate 141 (E141). shPARG HCT116 cells were treated with H2O2 (2 mM in PBS, 5 min) and ADP-ribosylated residues were tagged by a hydroxamic acid derivative with an addition of 15.0109 Da, an increment that can be readily distinguished by mass spectrometry. Fragmentation of the NH2OH-derivatized peptides yielded typical b- and y-ion series, allowing easy localization of ADP-ribosylation sites. Glutamate 141 (E141) is indicated by an asterisk. The E141A mutation abolishes the ADP-ribosylation of H2AX. Empty vector (EV), wild type (WT), or the E141A mutant (E141A) of H2AX was expressed in U2OS H2AX knockout cells that were treated with H2O2. ADP-ribosylated proteins were IPed with anti-ADPR antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody. ADP-ribosylation on H2AX is suppressed by olaparib treatment (1 µM, 1 h). 0.1% DMSO in medium for 1 h as mock. The samples were examined by IP and Western blotting. PARP1, but not PARP2, mediates the ADP-ribosylation on H2AX. Wild-type (WT) cells, PARP1-null cells, or PARP2-null cells were treated with H2O2. Source data are available online for this figure.Source data are available online for this figure. Source Data for Figure 1 [embj2020104542-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Next, to distinguish whether this modification is poly(ADP-ribosyl)ation (PolyADPR), oligo(ADP-ribosyl)ation (OligoADPR), or mono(ADP-ribosyl)ation (MonoADPR), the anti-ADP-ribose (anti-ADPR) antibody used in the tests can recognize pan-ADPR (Appendix Fig S3A). In addition, we purchased anti-pan-ADPR binding reagent from Millipore. Again, we identified the ADP-ribosylation of H2AX in response to oxidative damage (Appendix Fig S3B). Interestingly, with longer exposure, we observed smeared bands on ADP-ribosylated H2AX, indicating that the polymer form of ADP-ribosylation may exist on H2AX (Fig EV1A). Finally, we were able to use anti-PAR antibody to detect ADP-ribosylated H2AX, although the signals were relatively weak (Fig EV1B). Collectively, these results suggest that oxidative damage induces ADP-ribosylation on H2AX might be very short PAR chains or oligo(ADP-ribosyl)ation. Click here to expand this figure. Figure EV1. Oligo(ADP-ribose) or short PAR chains are synthesized on H2AX following DNA damage Smeared band of ADP-ribosylated H2AX was observed with longer exposure. 293T cells were treated with H2O2 (2 mM in PBS, 5 min), MMS (1 mM in medium, 30 min), or mock (PBS, 30 min). ADP-ribosylated proteins were immunoprecipitated with anti-ADPR antibody and examined by Western blotting using anti-H2AX antibody. 293T cells were treated with H2O2 (2 mM in PBS, 5 min), MMS (1 mM in medium, 30 min), or mock (PBS, 30 min). PARylated proteins were immunoprecipitated with anti-PAR antibody. PARylated-H2AX was examined by Western blotting using anti-H2AX antibody. Source data are available online for this figure. Download figure Download PowerPoint To exclude the cell line-specific ADP-ribosylation, we used HCT116 cells to further validate the ADP-ribosylation of H2AX. Since ADP-ribosylation is a reversible posttranslational modification and removal of PARylation is mainly mediated by PARG, knockdown PARG in HCT116 cells suppresses dePARylation (Leung, 2014; Kraus, 2015; Liu & Yu, 2015; Kassab et al, 2020). HCT116 shPARG cells were treated with H2O2 or MMS, and we found that H2AX was ADP-ribosylated using IP and Western blotting (Appendix Fig S4A), Additionally, to exclude off-targeted effects of knockdown PARG, we lysed the HCT116 cells with 0.5% SDS buffer which is sufficient to denature the enzymatic activity of PARG in cell lysates. Again, we still observed the ADP-ribosylation of H2AX (Appendix Fig S4B). Next, to explore the sites of ADP-ribosylation on H2AX, an unbiased high-resolution mass spectrometry was performed using HCT116 shPARG cell lysates treated with H2O2 (Zhang et al, 2013). The ADP-ribosylated residues were tagged by a hydroxamic acid derivative with an addition of 15.0109 Da, an increment that can be readily distinguished by mass spectrometry. Fragmentation of the NH2OH-derivatized peptides yielded typical b- and y-ion series, allowing easy localization of ADP-ribosylation sites. The results identified ADP-ribosylation of H2AX on glutamate 141 (E141), and these data have been reported by Dr. Yonghao Yu group in Nature Methods in 2013 (Fig 1B). To further validate the ADP-ribosylation at E141 in cells, CRISPR/Cas9 was used to delete H2AX in U2OS cells. The deficient cells were reconstituted with either empty vector (EV) or wild-type H2AX (WT) (Appendix Fig S5A). We also mutated the glutamate 141 into alanine (the E141A mutant) and reconstructed into H2AX knockout U2OS cells (Appendix Fig S5A). The expression levels of reconstituted wild-type H2AX (H2AX KO + WT) and the E141A mutant (H2AX KO + E141A) were very similar. We also compared and found the level of exogenous H2AX was very similar to that of endogenous H2AX (Appendix Fig S5B). Importantly, the E141A mutant was properly incorporated into the chromatin (Appendix Fig S5C). Unlike the wild-type H2AX, the E141A mutant abolished DNA damage-induced ADP-ribosylation (Fig 1C). Moreover, the DNA damage-induced ADP-ribosylation event on H2AX was also suppressed by PARP inhibitor (olaparib) treatment (Fig 1D). Since olaparib suppresses the enzymatic activities of PARP1 and PARP2, we further examined the ADP-ribosylation of H2AX in PARP1- and PARP2-deficient cells, and found that lack of PARP1 abolished DNA damage-induced ADP-ribosylation on H2AX (Fig 1E), suggesting that PARP1 mediates this ADP-ribosylation event in the cells. Collectively, these results suggest that the E141 of H2AX is ADP-ribosylated in response to DNA damage. The ADP-ribosylation at E141 of H2AX mediates BER Next, we explored the biological function of this ADP-ribosylation event. We treated cells with H2O2 or MMS and performed MTT assays to evaluate short-term cell viability. We found that cells lacking H2AX were hypersensitive to H2O2 or MMS treatment (Fig 2A), which is consistent with earlier studies (Revet et al, 2011). Interestingly, cells only expressing the E141A mutant were also hypersensitive to H2O2 or MMS treatment (Fig 2A). To further validate these results, we performed cell colony formation assays to examine long-term cell viability under similar genotoxic stress. Again, cells lacking H2AX or only expressing the E141A mutant were hypersensitive to the H2O2 or MMS treatment (Fig 2B). Figure 2. The ADP-ribosylation at E141 of H2AX mediates BER U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO + EV), wild-type H2AX (H2AX KO + WT), or the E141A mutant H2AX (E141A) were treated with H2O2 or MMS. Cell viability was measured after 24 h using MTT assay. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01. U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO + EV), wild-type H2AX (H2AX KO + WT), or the E141A mutant (E141A) H2AX were treated with H2O2 (2 mM in PBS, 5 min) or MMS (1 mM in medium, 30 min); subsequently, the cells were cultured in fresh DMEM for 2 weeks. The numbers of colony formation were counted. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01. Damaged base repair is suppressed in U2OS cells expressing the E141A mutant (E141A). U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with wild-type H2AX (H2AX KO + WT), or the E141A mutant H2AX (E141A) were treated with H2O2 (2 mM in PBS, 5 min) or MMS (1 mM in medium, 30 min) and harvested at the indicated recovery time points. Then, FPG-modified comet assay was performed. NT: no treatment. Representative comet tails were shown. The olive tail moments (OTM) were summarized from at least 50 cells in each experiment. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. N.S.: nonsignificant and **P < 0.01. Scale bar, 10 μm. DNA replication in U2OS cells expressing the E141A mutant (E141A) is suppressed. Cells were treated with H2O2 (2 mM in PBS, 5 min), MMS (1 mM in medium, 30 min), or mock (PBS, 30 min), followed by BrdU incorporation (30 μM in medium, 30 min). Representative images show BrdU-positive cells in cells expressing WT H2AX or E141A following genotoxic stress. Nuclei were stained with DAPI (blue). The bottom panel histogram shows the percentage of BrdU incorporation in each sample. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. ***P < 0.001. Scale bar, 30 μm. Generation of AP sites is suppressed in cells expressing E141A. Cells were treated with 2 mM H2O2 for 5 min or 1 mM MMS for 30 min. AP sites were calculated according to the standard curve. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. N.S.: nonsignificant and **P < 0.01. Download figure Download PowerPoint Both H2O2 and MMS induce various types of DNA base lesions as well as single-strand breaks (SSBs), which are mainly repaired by the base excision repair (BER) pathway. To study the role of ADP-ribosylation of H2AX in BER, we performed FPG-modified alkaline comet assay (Collins et al, 1993). Unexpectedly, we found accumulated DNA base damage in cells expressing the E141A mutant (Fig 2C), whereas no DSBs was detected following H2O2 treatment using neutral comet assay (Appendix Fig S6). To explore how the ADP-ribosylation of H2AX results in accumulation of DNA base damage, we examined and found that the replication recovery was remarkably delayed when cells were treated with H2O2 or MMS. These results indicate that a set of base lesions may not be processed effectively under these conditions, which may also block DNA replication (Fig 2D). To examine the role of H2AX ADP-ribosylation in BER, we explored the first step of BER, namely removal of damaged DNA base. Only if damaged bases are removed, BER will proceed and replication will resume. Once the damaged bases are removed from DNA lesions, apurinic/apyrimidinic (AP) sites will be generated. Thus, we measured the number of AP sites in genomic DNA (Appendix Fig S7). AP sites were reduced in cells only expressing the E141A mutant (Fig 2E), suggesting that base lesions may accumulate in these cells and ADP-ribosylation of H2AX may participate in the first step of BER. Taken together, these results suggest that ADP-ribosylation at E141 plays an important role in BER. ADP-ribosylation mediates the recruitment of Neil3 to DNA lesions Several glycosylases including OGG1 and Nei-like (Neil) family enzymes are known to recognize and excise damaged bases (Jacobs & Schar, 2012). Thus, we asked which enzyme could mediate H2AX-dependent ADP-ribosylation during BER, and examined the recruitment of these enzymes to DNA lesions using laser microirradiation assays. We observed that OGG1 was quickly recruited to DNA lesions. However, the recruitment of OGG1 was not affected by protein ADP-ribosylation, indicating that OGG1 does not mediate H2AX ADP-ribosylation-dependent BER (Appendix Fig S8). Interestingly, among three Neil family enzymes, Neil3 was quickly recruited to DNA lesions and the recruitment of Neil3 was abolished upon PARP inhibitor olaparib treatment (Fig 3A and Appendix Fig S9A and B), suggesting that DNA damage-induced ADP-ribosylation controls the recruitment of Neil3. In addition, PARG inhibitor treatment had no effect on the recruitment of Neil3 (Appendix Fig S10). Because Neil3 is a nuclear polypeptide with multiple domains, we generated a series of truncation mutants to map the domain that mediates the recruitment of Neil3. Notably, only the C-terminal domain GRF zinc-finger motifs of Neil3 (GRFs), but not other domains, was recruited to DNA lesions. Moreover, Neil3 lacking the GRFs could not be recruited, suggesting that the GRFs of Neil3 are necessary and sufficient for the relocation of Neil3 to DNA lesions (Fig 3B). In addition, PARP inhibitor treatment or lack of PARP1 suppressed the relocation of the GRFs (Fig 3C), further confirming that the recruitment of Neil3 to DNA lesions is ADP-ribosylation dependent. Since laser microirradiation generates a mixture of DNA lesions, including both DSBs and SSBs, we further validated the results using KillerRed system, which mainly induces oxidative DNA damage and BER. Consistently, Neil3 was recruited to the oxidative lesions and the recruitment was mediated by ADP-ribosylation (Fig 3D). To validate the Neil3 recruitment into DNA damage site is PARP1-dependent, we also examined the Neil3 recruitment in U2OS TRE PARP1 knockout cells. Again, our results showed that the relocation of Neil3 to the sites of DNA damage was impaired in the PARP1-deficient cells, demonstrating that Neil3's recruitment is dependent on the PARP1-mediated ADP-ribosylation (Appendix Fig S11). Figure 3. ADP-ribosylation mediates the recruitment of Neil3 to DNA lesions ADP-ribosylation mediates the recruitment of Neil3. GFP-Neil3 was expressed in U2OS cells, and cells were treated with olaparib (1 µM, 1 h) or mock (0.1% DMSO in medium, 1 h). Following laser microirradiation treatment, the recruitment of Neil3 was examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represent mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment. P-values were calculated using Student's t-test. ***P < 0.001. Scale bar, 2 μm. The GRFs of Neil3 alone are sufficient to be recruited to the sites of DNA damage. Truncated mutants of Neil3 were generated and fused with a GFP tag. Following laser microirradiation treatment, the recruitment of truncated mutants Neil3 was examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represent mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment. P-values were calculated using Student's t-test. ***P < 0.001. Scale bar, 2 μm. PARP inhibitor treatment (olaparib: 1 µM, 1 h) or lack of PARP1 suppresses the relocation of the GRFs of Neil3 to the DNA lesions. The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represent mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment. P-values were calculated using Student's t-test. ***P < 0.001. Scale bar, 2 μm. The recruitment of Neil3 to the oxidative lesions is inhibited by olaparib treatment (1 µM, 1 h). 0.1% DMSO in medium for 1 h as mock. Oxidative damage was induced by KillerRed (KR) system with the exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 min. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of Neil3 foci co-localized with KR was quantified. Mean value with SD is from 20 cells. P-values were calculated using Student's t-test. ***P < 0.001. Download figure Download PowerPoint Neil3 recognizes ADP-ribosylation of H2AX Next, we investigated whether Neil3 directly recognizes ADP-ribosylation. We generated recombinant full-length Neil3 and GRFs Neil3, and found that both the full-length Neil3 and GRFs Neil3 bind to PAR in vitro (Fig 4A). To determine whether Neil3 recognizes H2AX ADP-ribosylation in cells, we performed tandem co-immunoprecipitation (IP) and Western blotting assays, and found that Neil3 interacted with ADP-ribosylated H2AX but not with the E141A mutant (Fig 4B). Moreover, the GRFs of Neil3 are required for the interaction (Fig 4C and D). Figure 4. Neil3 recognizes ADP-ribosylation of H2AX A. The GRFs of Neil3 recognize PAR. The recombinant GST fusion proteins were incubated with PAR, and the protein–PAR complex was pulled down by glutathione agarose beads. Samples were serially diluted threefold, spotted onto the nitrocellulose membrane, and subjected to dot blotting using anti-PAR antibody (top panel). FL represents full-length Neil3, N-ter represents N-terminal Neil3 (1–300 aa), Mid represents middle Neil3 (300–404 aa), and GRFs represent C-terminal Neil3 GRFs domain (480–606 aa). Recombinant GST protein was used as the negative control (NC). The GST fusion proteins were also examined by the SDS–PAGE followed with Coomassie blue staining (middle panel). The relative PAR binding in the pull-down assay was summarized in the graph (bottom panel). Values are mean ± SD of three assays. B. Tandem co-immunoprecipitation and Western blotting assays demonstrate that Neil3 interacts with ADP-ribosylated H2AX but not the E141A mutant H2AX. C, D. The GRFs of Neil3 are sufficient and required for the interaction with ADP-ribosylated H2AX. Tandem co-immunoprecipitation and Western blotting assays were used as in Fig 4B. E, F. The relocation kinetic of full-length Neil3 or the GRFs of Neil3 to DNA lesions was analyzed. GFP-tagged full-length Neil3 or the GRFs of Neil3 was expressed in the indicated cells, and relo