APE2 Is Required for ATR-Chk1 Checkpoint Activation in Response to Oxidative Stress

APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress

Jeremy Willis1, Yogin Patel1, Barry L. Lentz, and Shan Yan2

Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223

Edited* by Raymond L. Erikson, Harvard University, Cambridge, MA, and approved May 15, 2013 (received for review January 22, 2013) The base excision repair pathway is largely responsible for the repair endonucleases (23, 24). APE2, which has weak AP endonuclease of oxidative stress-induced DNA damage. However, it remains unclear activity and strong 3′-phosphodiesterase and 3′-5′ exonuclease how the DNA damage checkpoint is activated by oxidative stress at activities, is a key player in PCNA-dependent repair of hydrogen the molecular level. Here, we provide evidence showing that hydro- peroxide (H2O2)-induced oxidative DNA damage (25–27). gen peroxide (H2O2) triggers checkpoint kinase 1 (Chk1) phosphoryla- However, the biological significance of APE2 in the DNA damage tion in an ATR [ataxia-telangiectasia mutated (ATM) and Rad3- response has not been elucidated. As far as we know, it remains related]-dependent but ATM-independent manner in Xenopus egg elusive whether the DNA damage checkpoint and BER pathways extracts. A base excision repair protein, Apurinic/apyrimidinic (AP) en- are coordinated in cellular responses to oxidative stress. donuclease 2 (APE2, APN2, or APEX2), is required for the generation of We performed a series of studies in checkpoint signaling using replication protein A (RPA)-bound single-stranded DNA, the recruit- Xenopus egg extracts, a well-characterized cell-free model system ment of a checkpoint protein complex [ATR, ATR-interacting protein (28–32). Here, we provide evidence that suggests APE2 is required (ATRIP), and Rad9] to damage sites, and H2O2-induced Chk1 phosphor- for ATR-Chk1 checkpoint activation in response to oxidative ylation. A conserved proliferating cell nuclear antigen interaction stress. Molecular characterization of the underlying mechanisms of protein box of APE2 is important for the recruitment of APE2 to APE2 has shed light on two distinct roles for APE2 in the ATR- ′ ′ ′ H2O2-damaged chromatin. APE2 3 -phosphodiesterase and 3 -5 Chk1 checkpoint: ssDNA generation via 3′-5′ SSB end resection exonuclease activity is essential for single-stranded DNA genera- and Claspin-like Chk1 binding. These roles of APE2 in the ATR- tion in the 3′–5′ direction from single-stranded breaks, referred to Chk1 checkpoint will help us better understand the DNA damage as single-stranded break end resection. In addition, APE2 associ- response after oxidative stress and provide a clear connection be- BIOCHEMISTRY ates with Chk1, and a serine residue (S86) in the Chk1-binding tween the BER pathway and DNA damage checkpoint signaling. motif of APE2 is essential for Chk1 phosphorylation, indicating a Claspin-like but distinct role for APE2 in ATR-Chk1 signaling. Results Our data indicate that APE2 plays a vital and previously unex- Hydrogen Peroxide Induces ATR-Dependent but ATM-Independent pected role in ATR-Chk1 checkpoint signaling in response to oxi- Chk1 Phosphorylation in Xenopus Egg Extracts. To study oxidative dative stress. Thus, our findings shed light on a distinct mechanism stress, we first tested whether H2O2 triggers a checkpoint response of how an ATR-Chk1–dependent DNA damage checkpoint is me- in Xenopus egg extracts. Chk1 phosphorylation at serine 344 (Chk1 diated by APE2 in the oxidative stress response. P-S344) is an indicator of ATR activation (33, 34). Although lower concentrations of H2O2 (1 and 10 mM) triggered relatively weak ells are constantly challenged by exogenous and endogenous Chk1 phosphorylation (Fig. 1A), we consistently observed Chk1 Cinsults that threaten genomic integrity. Excess accumulation P-S344 at 100 mM H2O2. Chk1 phosphorylation occurred at 40, 60, of reactive oxygen species leads to oxidative DNA damage, such and 80 min, but not at 20 min (Fig. 1B). These data suggest that as DNA strand breaks with 3′-modified termini, which is often the H2O2 induces a checkpoint response in a dose- and time-dependent underlying pathology in a variety of diseases including neurode- manner. ATM specific inhibitor KU55933 inhibited H2O2-induced generative diseases and cancer (1–6). Cellular responses to DNA ATM phosphorylation at serine 1981 (ATM P-S1981), but not Chk1 damage are mainly coordinated by two distinct DNA damage phosphorylation (Fig. 1C). In addition, both ATM phosphorylation checkpoint signaling cascades: ATM (ataxia-telangiectasia mu- and Chk1 phosphorylation were compromised by caffeine addition. tated)-checkpoint kinase 2 (Chk2) and ATR (ATM and Rad3- This evidence suggests that H2O2-induced Chk1 phosphoryla- related)-checkpoint kinase 1 (Chk1) pathways (7–10). ATM is tion is ATR-dependent, but ATM-independent. To directly test activated by intermolecular autophosphorylation and dimer dis- whether ATR is required for H2O2-induced Chk1 phosphorylation, sociation in response to double-stranded beaks (DSBs) (11–13). we removed ATR-interacting protein (ATRIP) from egg extract by ATR is activated by primed single-stranded DNA (ssDNA) in immunodepletion (Fig. 1D). ATRIP antibodies codepleted ATR, response to a variety of DNA damage or replication stresses (14, consistent with a previous study (35). H2O2-induced Chk1 phos- 15). Oxidative stress has been demonstrated to activate an ATM- phorylation was compromised in ATRIP-depleted egg extract (Fig. dependent DNA damage checkpoint (16–18). However, in pre- 1D). Both ATR and ATRIP were recruited to H2O2-damaged vious studies, hyperoxic conditions resulted in the phosphorylation chromatin in mock-depleted egg extracts, and as expected, ATR of Chk1 and p53 in an ATR-dependent but ATM-independent and ATRIP recruitment to H2O2-damaged chromatin was com- fashion (19). Furthermore, it remains unclear which specificDNA promised in ATRIP-depleted egg extracts (Fig. 1D). In addition, structures trigger checkpoint signaling during oxidative stress. H2O2 induced replication protein A 32 (RPA32) phosphorylation To eliminate oxidative DNA damage, base excision repair (BER) has evolved as a major DNA damage repair mechanism (20). In the initial step of BER, oxidatively damaged bases are excised by Author contributions: J.W., Y.P., and S.Y. designed research; J.W., Y.P., B.L.L., and S.Y. DNA glycosylases, generating apurinic/apyrimidinic (AP) sites. An performed research; J.W., Y.P., and S.Y. analyzed data; and J.W. and S.Y. wrote the paper. incision at the AP site by AP endonuclease or AP lyase generates The authors declare no conflict of interest. a single-stranded break (SSB) (21). Subsequently, the SSB is fixed *This Direct Submission article had a prearranged editor. via collaboration between proliferating cell nuclear antigen 1J.W. and Y.P. contributed equally to this work. (PCNA), DNA polymerase β, replication factor C, flap endonu- 2To whom correspondence should be addressed. E-mail: [email protected]. clease 1 (FEN1), and DNA ligase I (22). APE1 (AP endonuclease This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1) and APE2 (AP endonuclease 2) are the two characterized AP 1073/pnas.1301445110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1301445110 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 extract (Fig. 2D). Adding back recombinant Myc-APE2 to APE2- depleted egg extracts rescued H2O2-induced Chk1 phosphorylation (Fig. 2D). Therefore, we conclude that APE2 is required for H2O2- induced Chk1 phosphorylation. To test whether APE2 is required for Chk1 phosphorylation induced by stalled DNA replication forks, we investigated the effect of aphidicolin as a DNA replication stressor. As shown in Fig. 2D, APE2 was dispensable for aphidicolin-induced Chk1 phosphorylation, suggesting that the molecular mechanism of APE2 for the ATR-Chk1 checkpoint in oxidative stress is not tied to the uncoupling of minichromosome maintenance (MCM) helicase and DNA polymerase activities. The assembly of DNA damage checkpoint proteins at DNA breaks is vital to triggering checkpoint signaling (37). To better understand the underlying molecular mechanism, we reasoned that APE2 might be required for the recruitment of ATR and ATRIP to damaged chromatin. H2O2-induced recruitment of ATR and ATRIP was indeed compromised in APE2-depleted egg extracts (Fig. 2E). Replication protein A (RPA) binds to ssDNA and is required for ATRIP-mediated ATR recruitment to stalled replication forks or damage sites (38–40). RPA hyperloading to Fig. 1. Hydrogen peroxide triggers an ATR-dependent, but ATM-indepen- damaged chromatin indirectly indicates the generation of ssDNA dent, Chk1 phosphorylation in Xenopus egg extracts. (A) Sperm chromatin (41, 42). RPA32, the second largest subunit of the heterotrimeric was added to Xenopus egg extracts supplemented with different concen- RPA complex, was hyperloaded to H2O2-damaged chromatin, trations of H2O2, as indicated. Chk1 phosphorylation at S344 and total Chk1 indicating ssDNA generation after H O treatment (Fig. 2E were examined via immunoblotting. (B) Sperm chromatin was added to egg 2 2 and Fig. S1). More important, H2O2-induced hyperloading of extracts with the presence or absence of H2O2 (100 mM). At different times, as E indicated, samples were collected and examined as in A.(C) KU55933 or caf- RPA32 was compromised in APE2-depleted egg extracts (Fig. 2 ), feine was incubated with egg extracts, followed by the addition of sperm

chromatin and H2O2. Samples were examined for the indicated proteins. (D) Sperm chromatin was added to mock-depleted or ATRIP-depleted egg ex-

tracts supplemented with H2O2. Chromatin-bound fractions (“chromatin” panel) and total egg extracts (“extract” panel) were analyzed for the indicated proteins via immunoblotting.

at serine 33 (RPA32 P-S33) (Fig. S1), which also indicates ATR activation (36). Taken together, these data suggest that H2O2 triggers Chk1 phosphorylation in Xenopus egg extracts in an ATR- dependent but ATM-independent fashion.

APE2 Is Required for Chk1 Phosphorylation Induced by Hydrogen Peroxide but Not by Stalled Replication Forks. Domain dissection of APE2 shows three highly conserved domains: an AP endonuclease domain for its enzymatic activities on the N terminus (aa 2– 307), a zinc finger domain on the C terminus (aa 461–508), and a PCNA interacting protein (PIP) box (aa 395–402) (Fig. 2A). To study the role of APE2, we expressed and purified recombinant GST-APE2 protein from Escherichia coli, which was used for custom antibody production in rabbits (Fig. S2 A and B). Our anti-APE2 antibodies recognized endogenous APE2 (∼65 kD) and recombinant Myc-tagged-APE2 (Myc-APE2) (∼75 kD) and efficiently removed endogenous APE2 by immunodepletion from egg extracts (Fig. S2C). Notably, APE2 was preferentially recruited to H2O2-damaged chromatin at 40 min (Fig. 2B), suggesting a possible hit-and-run mechanism of APE2 recruitment to damage sites. It was also noticed that tiny APE2 was recruited to Fig. 2. APE2 preferentially binds to hydrogen peroxide-damaged chromatin chromatin at 20 and 80 min in unperturbed samples, which may and is required for hydrogen peroxide-induced Chk1 phosphorylation. (A) Schematic diagram of APE2. (B) Sperm chromatin was added to egg extracts suggest that a low amount of possible endogenous DNA damage “ ” supplemented with H2O2. At different times, chromatin-bound fractions and can be recognized by APE2 under normal conditions. However, total egg extracts were examined for the indicated proteins. (C) Recombinant this tiny recruitment of APE2 did not trigger Chk1 phosphory- Myc-APE2 was added to egg extracts supplemented with sperm chromatin and

lation (Fig. 1B), suggesting that a possible threshold of accumu- H2O2. Chromatin-bound fractions and total egg extracts were analyzed at 40 lated DNA damage may be needed for a robust checkpoint signal. min via immunoblotting. (D) Sperm chromatin was added to mock- or APE2- Similarly, recombinant Myc-APE2 also showed the preferential depleted egg extracts supplemented with recombinant Myc-APE2 and H2O2 ﬁ – binding to H O -damaged chromatin (Fig. 2C). or aphidicolin (APX), as indicated. *, nonspeci c bands in lanes 1 6andan 2 2 overlapping band of the nonspeciﬁc band and Myc-APE2 in lanes 7–9. Endo. To directly test the role of APE2 in DNA damage checkpoint APE2, endogenous APE2 in egg extracts. (E) Sperm chromatin was added to signaling, we added sperm chromatin to either mock- or APE2- mock- or APE2-depleted egg extracts supplemented with recombinant Myc-

depleted egg extracts supplemented with H2O2.H2O2-induced APE2 and H2O2, as indicated. Samples were examined for the indicated pro- Chk1 phosphorylation was compromised in APE2-depleted egg teins from chromatin fraction or extract.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1301445110 Willis et al. Downloaded by guest on September 23, 2021 suggesting APE2 is responsible for ssDNA generation. To activate ATR kinase, the 9-1-1 (Rad9-Rad1-Hus1) complex must be recruited to damage sites (43, 44). The Rad9 recruitment to H2O2- damaged chromatin was also compromised in APE2-depleted egg extracts (Fig. 2E). Adding back recombinant Myc-APE2 to APE2- depleted egg extracts rescued the recruitment of ATR, ATRIP, Rad9, and RPA32 to H2O2-damaged chromatin and H2O2- induced Chk1 phosphorylation (Fig. 2E). These data suggest that APE2 is essential for the generation of RPA-bound ssDNA on damaged chromatin, thereby contributing to a checkpoint protein complex assembly onto H2O2-damaged sites. This ﬁnding reveals a distinct molecular mechanism of how APE2 contributes to ATR activation.

PCNA Association and Enzymatic Activities of APE2 Are Required for Chk1 Phosphorylation in Response to Hydrogen Peroxide. PCNA, a DNA sliding clamp complex, orchestrates a variety of crucial players to replication forks (45, 46). Previously, it was established that APE2 associates with PCNA via a PIP box of APE2 in humans and yeast (25, 26, 47). To directly assess the role of APE2’s binding to PCNA, we constructed a PIP box mutant of Myc-APE2 with mutations from phenylalanine to alanine at amino acid residues 401 and 402 (FF; Fig. 3A). The wild-type Myc-APE2 is designated as WT. A coimmunoprecipitation experiment with anti-PCNA antibodies in APE2-depleted egg extracts showed that anti-PCNA antibodies immunoprecipitated WT but not FF APE2, indicating the PIP box is important for APE2 binding to PCNA (Fig. S3B). In

APE2-depleted egg extracts (Fig. S3A), exogenous WT APE2, but BIOCHEMISTRY not FF APE2, efficiently associated with H2O2-damaged chromatin when added at a similar level (Fig. 3B). Consistent with this result, FF APE2 failed to rescue the recruitment of RPA32 and the checkpoint complex (ATR, ATRIP, and Rad9) onto H2O2- damaged chromatin (Fig. 3B). Moreover, FF APE2 did not rescue H2O2-induced Chk1 phosphorylation in APE2-depleted egg extracts (Fig. 3C). In addition, the control experiment has shown that other APE2 mutants in this study have no deficiency in PCNA association (Fig. S3C). These observations indicate that the PCNA association with APE2 via the PIP box region is important for the recruitment of APE2 to H2O2-damaged chromatin, RPA recruitment to ssDNA, assembly of the checkpoint protein complex Fig. 3. Critical role of PCNA association and enzyme activities of APE2 in on damaged chromatin, and subsequent Chk1 phosphorylation. response to hydrogen peroxide-induced damage. (A) Schematic diagram for Previous intensive studies have demonstrated that budding WT, PIP box mutant (FF), enzyme activity mutants (EA and DA), and CKB yeast Apn2 (the yeast homologue of the APE2) with glutamic acid mutant (SA) of Myc-APE2. (B) WT or FF APE2 was added to APE2-depleted 59 mutation to alanine and human APE2 with aspartate 277 egg extracts supplemented with sperm chromatin and H2O2. Chromatin mutation to alanine are defective in its 3′-phosphodiesterase and fractions and total extracts were examined for the indicated proteins. (C) 3′-5′ exonuclease activities (48, 49). These two critical residues in Chk1 phosphorylation was analyzed from the samples in B.(D) WT, EA, or APE2 are identical in frog, budding yeast, fission yeast, mouse, DA APE2 was added to APE2-depleted egg extracts supplemented with sperm chromatin and H2O2. Chromatin fractions and total extracts were and human (Fig. S4), suggesting both residues may be in the ac- analyzed for the indicated proteins. (E) Chk1 phosphorylation was examined tive site of APE2 enzyme and very conserved for its enzymatic from the samples in D. activity during evolution. To test whether APE2’s enzyme activity is essential for checkpoint signaling, we constructed two mutant Xenopus Myc-APE2 with a glutamic acid to alanine mutation at APE2 Associates with Chk1. Finally, we considered a possible as- residue 34 or an aspartate to alanine mutation at residue 273 (EA sociation between APE2 and Chk1. We tested this hypothesis and DA; Fig. 3A). Consistent with the assumption that EA and through three lines of experiments. First, we added recombinant DA APE2 have enzymatic activity deficiency, we found that nei- Chk1-GH protein (Chk1 with GST-tags and His-tags) to egg ther of them rescued RPA32 hyperloading and the efficient ATR extracts supplemented with sperm chromatin and H2O2. After checkpoint protein complex assembly to H2O2-damaged chro- washing, we examined the bead-bound fractions for APE2 and matin in APE2-depleted egg extracts, although EA and DA Chk1 via immunoblotting. APE2 can bind to Chk1-coupled APE2 could bind to H O -damaged chromatin efficiently (Fig. 2 2 nickel-nitrilotriacetic acid (Ni-NTA) beads. Notably, the addi- 3D). Moreover, neither EA APE2 nor DA APE2 rescued H2O2- tion of sperm chromatin and H2O2 has no noticeable effect on induced Chk1 phosphorylation in APE2-depleted egg extracts A (Fig. 3E). These results suggest that the end processing of the SSB the binding of APE2 to Chk1 (Fig. 4 ), suggesting that the as- 3′-termini by APE2’s3′-phosphodiesterase and 3′-5′ exonuclease sociation between APE2 and Chk1 may be constitutive. The activities may generate a long stretch of ssDNA, thereby creating control experiment has shown that APE2 was observed in Chk1- the structure for RPA binding followed by assembly of the GH-coupled bead-bound chromatin fraction, but not in “No checkpoint complex. We propose to refer to this phenomenon as Chk1-GH” controls (Fig. S3D). Second, we performed GST-pull- 3′-5′ SSB end resection, borrowing a term from a similar process down assays and showed that GST-APE2, but not GST or no of DSB end resection in a 5′-3′ direction (50). addition (−), can pull down endogenous Chk1 from egg extracts

Willis et al. PNAS Early Edition | 3of6 Downloaded by guest on September 23, 2021 two short conserved repeats of 10 amino acids were identified and characterized as Chk1-binding motifs (CKB motifs): ExxxLC (S/T)GxF (x represents any amino acid) (53). In Xenopus APE2, we found a Claspin-like CKB motif in the N-terminal domain: EEGLSGVF. Five amino acids in this APE2 motif are identical to the two conserved CKB motifs of Claspin (Fig. 4D). Notably, S86 of APE2 is identical to S864 and S895 of Claspin, whose phosphorylation is vital for binding to Chk1 and following Chk1 phosphorylation (Fig. 4D) (53). In addition, the CKB motif of APE2 is highly conserved in Xenopus, human, mouse, budding yeast, and fission yeast, with either serine or threonine in the fifth position of the CKB motif (Fig. 4E). We then tested whether the S86 of APE2 is essential for Chk1 binding. Our protein–protein interaction assays show that recombinant Chk1-GH associated with WT APE2, but not a mutant of APE2 from serine to alanine at residue 86 (SA; Figs. 3A and 4F). More significant, H2O2- induced Chk1 phosphorylation could be rescued by WT APE2, but not by SA APE2, in APE2-depleted extracts, although both WT and SA APE2 were efficiently recruited to H2O2-damaged chromatin (Fig. 4G). We also noticed that RPA32 was hyperloaded to H2O2-damaged chromatin and phosphorylated at serine 33 (RPA32 P-S33) after WT APE2 or SA APE2 was added back to APE2-depleted egg extracts (Fig. 4G). The control experiment has shown that other APE2 mutants including

Fig. 4. Binding of Chk1 to APE2 is required for Chk1 phosphorylation by activated ATR. (A) Recombinant Chk1-GH (Chk1 with GST-tags and His-tags)

was added to egg extracts supplemented with sperm chromatin and H2O2 as indicated. After incubation, extracts were mixed with nickel-nitrilotriacetic acid (Ni-NTA) beads. After washing, total egg extracts (Input, 5%) and bead- bound fractions were examined via immunoblotting. (B) GST or GST-APE2 was added to egg extracts supplemented with glutathione beads. Samples from GST pull-down and total egg extracts (Input) were examined for the indicated proteins. (C) GST or GST-APE2 was added to a binding buffer containing Ni-NTA beads coupled with Chk1-ΔKD-TH (a kinase deletion mutant of Chk1 with T7-tages and His-tags). The recombinant proteins in binding buffer (Input) and bead-bound fractions were examined via immunoblotting, as indicated. (D) Amino acid alignment of the CKB motifs in Xenopus APE2 and Claspin. In particular, S86 of APE2, S864 of Claspin, and S895 of Claspin are bolded and underlined. The CKB motifs are shown within the dashed rectangle. -, gaps in the alignment; *, identical residues; :,highly conserved residues; .,moderately conserved residues. (E) APE2 or its ortho- logs are aligned in Xenopus (Xe APE2), human (Hs APE2), mice (Ms APE2), ﬁssion yeast (Sp APN2), and budding yeast (Sc APN2). (F) WT or SA APE2 was added to egg extracts supplemented with Chk1-GH-coupled Ni-NTA beads. After a 1-h incubation, bead-bound and input were examined for the indicated proteins. (G) WT or SA APE2 was added to APE2-depleted egg

extracts supplemented with sperm chromatin and H2O2. Chromatin fractions and total extracts were examined for the indicated proteins.

(Fig. 4B). Finally, we tested the direct interaction between APE2 and Chk1 in vitro through a protein–protein interaction assay in which we added either GST or GST-APE2 to binding buffer containing Chk1-ΔKD-TH (a kinase domain deletion mutant of Chk1 with T7-tags and His-tags). Chk1-ΔKD-TH can pull down GST-APE2, but not GST (Fig. 4C), indicating a direct interaction between APE2 and Chk1. Thus, we conclude that APE2 associates with Chk1. Claspin, a vital Chk1-binding protein, plays an essential role in ATR-Chk1 checkpoint signaling in response to a variety of dif- Fig. 5. A model for APE2 in ATR-Chk1 checkpoint activation in the response ferent DNA damage and replication stress (51, 52). In Xenopus, to oxidative stress.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1301445110 Willis et al. Downloaded by guest on September 23, 2021 EA, DA, and FF APE2 characterized in this study are efficient in (CtIP), exonuclease 1 (Exo1), and RecJ exonuclease (50, 61, 62). Chk1 binding (Fig. S3E). These data suggest that the CKB motif Our study shows a distinct mechanism for ssDNA generation: 3′- of APE2 is important for activated ATR to phosphorylate Chk1, 5′ SSB end resection mediated by APE2. In addition, APE2 also but not other substrates of ATR, such as RPA32. Therefore, our makes Chk1 available to convey the signal from ATR to Chk1 study suggests another regulatory mechanism of the ATR-Chk1 when ATR is activated. Further study is needed to figure out checkpoint in oxidative stress: Claspin-like Chk1-binding of whether the S86 residue of APE2 is phosphorylated in response APE2 plays an indispensable role in Chk1 phosphorylation by to oxidative stress. We speculate that the mechanism of APE2 activated ATR kinase. in Chk1-binding might be distinct from Claspin, although they contain the same serine residue in the CKB motif. The 9-1-1 Discussion complex promotes BER via associating with several BER pro- Recent studies suggest that the mismatch repair and nucleotide teins including DNA glycosylases, polymerase β, APE1, FEN1, excision repair pathways contribute to checkpoint signaling through and DNA ligase I (63–69). However, it is unclear how the 9-1-1 is – DNA end-processing or by direct protein protein interactions recruited to damage sites. Our study provides a clue: the re- (54–56). However, there is very little evidence that shows the in- cruitment of the 9-1-1 to H2O2-damaged sites requires APE2- terplay between the BER pathway and the ATR checkpoint. Our mediated generation of RPA-bound ssDNA. Through positive data suggest that APE2 links the BER pathway to checkpoint feedback, the 9-1-1 then facilitates the BER pathway. Inter- signaling via a distinct mechanism. It has been suggested that estingly, APE2 has been proposed recently as a new disease enzymes containing 3′-5′ exonuclease activities are directly in- candidate gene for mitochondrial DNA maintenance disorders volved in maintaining genome stability (57). It has also been shown (70). In summary, our findings demonstrate a previously unex- that ssDNA gaps generated by DNA exonuclease III trigger an pected but essential role of APE2 in maintaining genomic sta- ATR- and cell division cycle 7 (Cdc7)- dependent checkpoint (58). Xenopus In this report, we identify and characterize APE2 as a distinct bility in cellular responses to oxidative stress in . checkpoint protein. We propose a model for APE2 in the ATR- Materials and Methods Chk1 checkpoint in response to oxidative stress (Fig. 5): step 1, an AP site is generated by oxidative stress; step 2, the AP site is in- The use and care of Xenopus laevis were approved by the Institutional cised to generate a SSB with a damaged 3′-terminus (such as 3′- Animal Care and Use Committee of the University of North Carolina at ′ Charlotte. Xenopus egg extracts were prepared as described previously (28, phosphate); step 3, PCNA is recruited to 3 -ssDNA/dsDNA 30). Sperm chromatin was obtained through previously described methods junction; step 4, together with Chk1, APE2 is recruited to damage BIOCHEMISTRY ′ (71). Sperm chromatin was added to egg extracts at a concentration of sites by PCNA via the PIP box; step 5, APE2 removes 3 -damaged ∼4,000 sperm/μL, and chromatin fractions were isolated as previously de- terminus, using its 3′-phosphodiesterase activity, and continues scribed (29). ATM inhibitor KU55933 (Calbiochem) was added to a final excision by its 3′-5′ exonuclease activity, thereby generating a long concentration of 120 μM, and PI3K inhibitor caffeine was added to 1 ng/μLas stretch of ssDNA, referred as 3′-5′ SSB end resection; step 6, previously described (72, 73). Hydrogen peroxide was added at the desig- a checkpoint protein complex including ATR, ATRIP and the 9-1- nated final concentrations to egg extract. Aphidicolin was added to a final 1 complex is assembled onto the RPA-bound ssDNA and con- concentration of 100 ng/μL (29). For immunodepletions, APE2 and ATRIP nected via topoisomerase II binding protein 1 (TopBP1), leading antibodies were used in a similar way as the TopBP1 immunodepletion to ATR activation; and step 7, activated ATR phosphorylates procedure (29). Chk1 while the 9-1-1 complex stimulates the BER pathway as Recombinant proteins, antibodies, and protein–protein interaction assays a positive feedback mechanism. are described in SI Materials and Methods. Our findings from this study provide insight into the molecular ACKNOWLEDGMENTS. We thank Dr. W. Matthew Michael for generous gifts mechanisms of RPA-bound ssDNA generation in ATR-Chk1 and critical comments on the manuscript; Drs. T. Slenn, L. Corey, and checkpoint signaling. In response to stalled DNA replication, the J. Weller for critical reading of the manuscript; Drs. K. Cimprich, W. G. Dunphy, RPA-bound ssDNA is generated via the functional uncoupling of H. Lindsay, and Z. You for reagents; and Drs. N. Lefebvre, C. D. Williams, and MCM helicase and DNA polymerase activities (14, 59, 60). In Y. M. Huet and the University of North Carolina at Charlotte Vivarium staff response to DSBs, the ATR kinase is activated after ATM ac- for maintaining the health of our frogs. This study was supported in part by ′ ′ funds provided by University of North Carolina at Charlotte and National tivation via the 5 -3 DSB end resection by several critical factors Institute of General Medical Sciences/National Institutes of Health Grant R15 such as C-terminal binding protein (CtBP) interacting protein GM101571 (to S.Y.).

1. Klaunig JE, Kamendulis LM, Hocevar BA (2010) Oxidative stress and oxidative damage 13. Daniel JA, et al. (2012) Loss of ATM kinase activity leads to embryonic lethality in in carcinogenesis. Toxicol Pathol 38(1):96–109. mice. J Cell Biol 198(3):295–304. 2. David SS, O’Shea VL, Kundu S (2007) Base-excision repair of oxidative DNA damage. 14. Cimprich KA, Cortez D (2008) ATR: An essential regulator of genome integrity. Nat Nature 447(7147):941–950. Rev Mol Cell Biol 9(8):616–627. 3. Odell ID, Wallace SS, Pederson DS (2013) Rules of engagement for base excision repair 15. Ciccia A, Elledge SJ (2010) The DNA damage response: Making it safe to play with – in chromatin. J Cell Physiol 228(2):258–266. knives. Mol Cell 40(2):179 204. 4. Friedberg EC (2003) DNA damage and repair. Nature 421(6921):436–440. 16. Xie S, et al. (2001) Reactive oxygen species-induced phosphorylation of p53 5. Sancar A, Lindsey-Boltz LA, Unsal-Kaçmaz K, Linn S (2004) Molecular mechanisms of on serine 20 is mediated in part by polo-like kinase-3. J Biol Chem 276(39): – mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73: 36194 36199. 17. Shackelford RE, et al. (2001) The Ataxia telangiectasia gene product is required for 39–85. oxidative stress-induced G1 and G2 checkpoint function in human ﬁbroblasts. J Biol 6. Hamdan SM, Richardson CC (2009) Motors, switches, and contacts in the replisome. Chem 276(24):21951–21959. Annu Rev Biochem 78:205–243. 18. Guo Z, Kozlov S, Lavin MF, Person MD, Paull TT (2010) ATM activation by oxidative 7. Harper JW, Elledge SJ (2007) The DNA damage response: Ten years after. Mol Cell 28 stress. Science 330(6003):517–521. (5):739–745. 19. Kulkarni A, Das KC (2008) Differential roles of ATR and ATM in p53, Chk1, and histone 8. Harrison JC, Haber JE (2006) Surviving the breakup: The DNA damage checkpoint. H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation. Am – Annu Rev Genet 40:209 235. J Physiol Lung Cell Mol Physiol 294(5):L998–L1006. 9. Branzei D, Foiani M (2010) Maintaining genome stability at the replication fork. Nat 20. Meira LB, Burgis NE, Samson LD (2005) Base excision repair. Adv Exp Med Biol 570: – Rev Mol Cell Biol 11(3):208 219. 125–173. 10. Bartek J, Lukas C, Lukas J (2004) Checking on DNA damage in S phase. Nat Rev Mol 21. Kim YJ, Wilson DM, 3rd (2012) Overview of base excision repair biochemistry. Curr Cell Biol 5(10):792–804. Mol Pharmacol 5(1):3–13. 11. Bakkenist CJ, Kastan MB (2003) DNA damage activates ATM through intermolecular 22. Hegde ML, Hazra TK, Mitra S (2008) Early steps in the DNA base excision/single-strand autophosphorylation and dimer dissociation. Nature 421(6922):499–506. interruption repair pathway in mammalian cells. Cell Res 18(1):27–47. 12. Lee JH, Paull TT (2005) ATM activation by DNA double-strand breaks through the 23. Tell G, Quadrifoglio F, Tiribelli C, Kelley MR (2009) The many functions of APE1/Ref-1: Mre11-Rad50-Nbs1 complex. Science 308(5721):551–554. Not only a DNA repair enzyme. Antioxid Redox Signal 11(3):601–620.

Willis et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 24. Boiteux S, Guillet M (2004) Abasic sites in DNA: Repair and biological consequences in 50. Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway Saccharomyces cerevisiae. DNA Repair (Amst) 3(1):1–12. choice. Annu Rev Genet 45:247–271. 25. Burkovics P, Hajdú I, Szukacsov V, Unk I, Haracska L (2009) Role of PCNA-dependent 51. Kumagai A, Dunphy WG (2000) Claspin, a novel protein required for the activation of stimulation of 3′-phosphodiesterase and 3′-5′ exonuclease activities of human Ape2 Chk1 during a DNA replication checkpoint response in Xenopus egg extracts. Mol Cell in repair of oxidative DNA damage. Nucleic Acids Res 37(13):4247–4255. 6(4):839–849. 26. Unk I, et al. (2002) Stimulation of 3′—>5′ exonuclease and 3′-phosphodiesterase ac- 52. Yoo HY, Jeong SY, Dunphy WG (2006) Site-specific phosphorylation of a checkpoint tivities of yeast apn2 by proliferating cell nuclear antigen. Mol Cell Biol 22(18): mediator protein controls its responses to different DNA structures. Genes Dev 20(7): – 6480 6486. 772–783. 27. Hadi MZ, Wilson DM, 3rd (2000) Second human protein with homology to the Es- 53. Kumagai A, Dunphy WG (2003) Repeated phosphopeptide motifs in Claspin mediate cherichia coli abasic endonuclease exonuclease III. Environ Mol Mutagen 36(4): the regulated binding of Chk1. Nat Cell Biol 5(2):161–165. – 312 324. 54. Brown KD, et al. (2003) The mismatch repair system is required for S-phase checkpoint 28. Yan S, Lindsay HD, Michael WM (2006) Direct requirement for Xmus101 in ATR-me- activation. Nat Genet 33(1):80–84. diated phosphorylation of Claspin bound Chk1 during checkpoint signaling. J Cell Biol 55. Yoshioka K, Yoshioka Y, Hsieh P (2006) ATR kinase activation mediated by MutSalpha 173(2):181–186. and MutLalpha in response to cytotoxic O6-methylguanine adducts. Mol Cell 22(4): 29. Yan S, Michael WM (2009) TopBP1 and DNA polymerase-alpha directly recruit the 9-1- 501–510. 1 complex to stalled DNA replication forks. J Cell Biol 184(6):793–804. 56. Giannattasio M, et al. (2010) Exo1 competes with repair synthesis, converts NER in- 30. Willis J, DeStephanis D, Patel Y, Gowda V, Yan S (2012) Study of the DNA damage termediates to long ssDNA gaps, and promotes checkpoint activation. Mol Cell 40(1): checkpoint using Xenopus egg extracts. J Vis Exp (69):e4449. 50–62. 31. Van C, Yan S, Michael WM, Waga S, Cimprich KA (2010) Continued primer synthesis at 57. Shevelev IV, Hübscher U (2002) The 3′ 5′ exonucleases. Nat Rev Mol Cell Biol 3(5): stalled replication forks contributes to checkpoint activation. J Cell Biol 189(2): – 233–246. 364 376. 32. Yan S, Willis J (2013) WD40-repeat protein WDR18 collaborates with TopBP1 to fa- 58. Costanzo V, et al. (2003) An ATR- and Cdc7-dependent DNA damage checkpoint that – cilitate DNA damage checkpoint signaling. Biochem Biophys Res Commun 431(3): inhibits initiation of DNA replication. Mol Cell 11(1):203 213. 466–471. 59. Byun TS, Pacek M, Yee MC, Walter JC, Cimprich KA (2005) Functional uncoupling of 33. Guo Z, Kumagai A, Wang SX, Dunphy WG (2000) Requirement for Atr in phosphor- MCM helicase and DNA polymerase activities activates the ATR-dependent check- – ylation of Chk1 and cell cycle regulation in response to DNA replication blocks and point. Genes Dev 19(9):1040 1052. UV-damaged DNA in Xenopus egg extracts. Genes Dev 14(21):2745–2756. 60. Lyubimov AY, Costa A, Bleichert F, Botchan MR, Berger JM (2012) ATP-dependent 34. Zhao H, Piwnica-Worms H (2001) ATR-mediated checkpoint pathways regulate conformational dynamics underlie the functional asymmetry of the replicative heli- phosphorylation and activation of human Chk1. Mol Cell Biol 21(13):4129–4139. case from a minimalist eukaryote. Proc Natl Acad Sci USA 109(30):11999–12004. 35. Kumagai A, Kim SM, Dunphy WG (2004) Claspin and the activated form of ATR-ATRIP 61. Shiotani B, Zou L (2009) Single-stranded DNA orchestrates an ATM-to-ATR switch at collaborate in the activation of Chk1. J Biol Chem 279(48):49599–49608. DNA breaks. Mol Cell 33(5):547–558. 36. Olson E, Nievera CJ, Klimovich V, Fanning E, Wu X (2006) RPA2 is a direct downstream 62. Handa N, Morimatsu K, Lovett ST, Kowalczykowski SC (2009) Reconstitution of initial target for ATR to regulate the S-phase checkpoint. J Biol Chem 281(51):39517–39533. steps of dsDNA break repair by the RecF pathway of E. coli. Genes Dev 23(10): 37. Polo SE, Jackson SP (2011) Dynamics of DNA damage response proteins at DNA 1234–1245. breaks: A focus on protein modifications. Genes Dev 25(5):409–433. 63. Chang DY, Lu AL (2005) Interaction of checkpoint proteins Hus1/Rad1/Rad9 with DNA 38. Zou L, Elledge SJ (2003) Sensing DNA damage through ATRIP recognition of RPA- base excision repair enzyme MutY homolog in fission yeast, Schizosaccharomyces ssDNA complexes. Science 300(5625):1542–1548. pombe. J Biol Chem 280(1):408–417. 39. Broderick S, Rehmet K, Concannon C, Nasheuer HP (2010) Eukaryotic single-stranded 64. Gembka A, et al. (2007) The checkpoint clamp, Rad9-Rad1-Hus1 complex, preferen- DNA binding proteins: Central factors in genome stability. Subcell Biochem 50: tially stimulates the activity of apurinic/apyrimidinic endonuclease 1 and DNA poly- – 143 163. merase beta in long patch base excision repair. Nucleic Acids Res 35(8):2596–2608. 40. Choi JH, et al. (2010) Reconstitution of RPA-covered single-stranded DNA-activated 65. Smirnova E, Toueille M, Markkanen E, Hübscher U (2005) The human checkpoint – ATR-Chk1 signaling. Proc Natl Acad Sci USA 107(31):13660 13665. sensor and alternative DNA clamp Rad9-Rad1-Hus1 modulates the activity of DNA 41. Fanning E, Klimovich V, Nager AR (2006) A dynamic model for replication protein A ligase I, a component of the long-patch base excision repair machinery. Biochem J 389 (RPA) function in DNA processing pathways. Nucleic Acids Res 34(15):4126–4137. (Pt 1):13–17. 42. Wold MS (1997) Replication protein A: A heterotrimeric, single-stranded DNA-binding 66. Friedrich-Heineken E, et al. (2005) The two DNA clamps Rad9/Rad1/Hus1 complex and protein required for eukaryotic DNA metabolism. Annu Rev Biochem 66:61–92. proliferating cell nuclear antigen differentially regulate flap endonuclease 1 activity. 43. Parrilla-Castellar ER, Arlander SJ, Karnitz L (2004) Dial 9-1-1 for DNA damage: The J Mol Biol 353(5):980–989. Rad9-Hus1-Rad1 (9-1-1) clamp complex. DNA Repair (Amst) 3(8-9):1009–1014. 67. Wang W, Lindsey-Boltz LA, Sancar A, Bambara RA (2006) Mechanism of stimulation of 44. Navadgi-Patil VM, Burgers PM (2009) A tale of two tails: Activation of DNA damage human DNA ligase I by the Rad9-rad1-Hus1 checkpoint complex. J Biol Chem 281(30): checkpoint kinase Mec1/ATR by the 9-1-1 clamp and by Dpb11/TopBP1. DNA Repair – (Amst) 8(9):996–1003. 20865 20872. 45. Moldovan GL, Pfander B, Jentsch S (2007) PCNA, the maestro of the replication fork. 68. Guan X, et al. (2007) The human checkpoint sensor Rad9-Rad1-Hus1 interacts with – Cell 129(4):665–679. and stimulates NEIL1 glycosylase. Nucleic Acids Res 35(8):2463 2472. ’ 46. Kelch BA, Makino DL, O’Donnell M, Kuriyan J (2011) How a DNA polymerase clamp 69. Eichinger CS, Jentsch S (2011) 9-1-1: PCNA s specialized cousin. Trends Biochem Sci 36 – loader opens a sliding clamp. Science 334(6063):1675–1680. (11):563 568. fi 47. Tsuchimoto D, et al. (2001) Human APE2 protein is mostly localized in the nuclei and 70. Wang W, et al. (2011) Identi cation of rare DNA variants in mitochondrial disorders – to some extent in the mitochondria, while nuclear APE2 is partly associated with with improved array-based sequencing. Nucleic Acids Res 39(1):44 58. proliferating cell nuclear antigen. Nucleic Acids Res 29(11):2349–2360. 71. Tutter AV, Walter JC (2006) Chromosomal DNA replication in a soluble cell-free sys- 48. Burkovics P, Szukacsov V, Unk I, Haracska L (2006) Human Ape2 protein has a 3′-5′ tem derived from Xenopus eggs. Methods Mol Biol 322:121–137. exonuclease activity that acts preferentially on mismatched base pairs. Nucleic Acids 72. Sarkaria JN, et al. (1999) Inhibition of ATM and ATR kinase activities by the radio- Res 34(9):2508–2515. sensitizing agent, caffeine. Cancer Res 59(17):4375–4382. 49. Unk I, Haracska L, Prakash S, Prakash L (2001) 3′-phosphodiesterase and 3′—>5′ 73. Hickson I, et al. (2004) Identification and characterization of a novel and specificin- exonuclease activities of yeast Apn2 protein and requirement of these activities for hibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res 64(24): repair of oxidative DNA damage. Mol Cell Biol 21(5):1656–1661. 9152–9159.

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