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 replica- tion 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