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. 2013 Jun 25;110(26):10592-7.
doi: 10.1073/pnas.1301445110. Epub 2013 Jun 10.

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

Jeremy Willis  1 , Yogin Patel, Barry L Lentz, Shan Yan
Affiliations

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

Jeremy Willis et al. Proc Natl Acad Sci U S A. .

Abstract

The base excision repair pathway is largely responsible for the repair of oxidative stress-induced DNA damage. However, it remains unclear how the DNA damage checkpoint is activated by oxidative stress at the molecular level. Here, we provide evidence showing that hydrogen peroxide (H2O2) triggers checkpoint kinase 1 (Chk1) phosphorylation in an ATR [ataxia-telangiectasia mutated (ATM) and Rad3-related]-dependent but ATM-independent manner in Xenopus egg extracts. A base excision repair protein, Apurinic/apyrimidinic (AP) endonuclease 2 (APE2, APN2, or APEX2), is required for the generation of replication protein A (RPA)-bound single-stranded DNA, the recruitment of a checkpoint protein complex [ATR, ATR-interacting protein (ATRIP), and Rad9] to damage sites, and H2O2-induced Chk1 phosphorylation. A conserved proliferating cell nuclear antigen interaction protein box of APE2 is important for the recruitment of APE2 to H2O2-damaged chromatin. APE2 3'-phosphodiesterase and 3'-5' exonuclease activity is essential for single-stranded DNA generation in the 3'-5' direction from single-stranded breaks, referred to as single-stranded break end resection. In addition, APE2 associates with Chk1, and a serine residue (S86) in the Chk1-binding motif of APE2 is essential for Chk1 phosphorylation, indicating a Claspin-like but distinct role for APE2 in ATR-Chk1 signaling. Our data indicate that APE2 plays a vital and previously unexpected role in ATR-Chk1 checkpoint signaling in response to oxidative stress. Thus, our findings shed light on a distinct mechanism of how an ATR-Chk1-dependent DNA damage checkpoint is mediated by APE2 in the oxidative stress response.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Hydrogen peroxide triggers an ATR-dependent, but ATM-independent, Chk1 phosphorylation in Xenopus egg extracts. (A) Sperm chromatin was added to Xenopus egg extracts supplemented with different concentrations of H2O2, as indicated. Chk1 phosphorylation at S344 and total Chk1 were examined via immunoblotting. (B) Sperm chromatin was added to egg extracts with the presence or absence of H2O2 (100 mM). At different times, as indicated, samples were collected and examined as in A. (C) KU55933 or caffeine 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 extracts supplemented with H2O2. Chromatin-bound fractions (“chromatin” panel) and total egg extracts (“extract” panel) were analyzed for the indicated proteins via immunoblotting.
Fig. 2.
Fig. 2.
APE2 preferentially binds to hydrogen peroxide-damaged chromatin and is required for hydrogen peroxide-induced Chk1 phosphorylation. (A) Schematic diagram of APE2. (B) Sperm chromatin was added to egg extracts supplemented with H2O2. At different times, chromatin-bound fractions and total egg extracts were examined for the indicated proteins. (C) Recombinant Myc-APE2 was added to egg extracts supplemented with sperm chromatin and H2O2. Chromatin-bound fractions and total egg extracts were analyzed at 40 min via immunoblotting. (D) Sperm chromatin was added to mock- or APE2-depleted egg extracts supplemented with recombinant Myc-APE2 and H2O2 or aphidicolin (APX), as indicated. *, nonspecific bands in lanes 1–6 and an overlapping band of the nonspecific band and Myc-APE2 in lanes 7–9. Endo. APE2, endogenous APE2 in egg extracts. (E) Sperm chromatin was added to mock- or APE2-depleted egg extracts supplemented with recombinant Myc-APE2 and H2O2, as indicated. Samples were examined for the indicated proteins from chromatin fraction or extract.
Fig. 3.
Fig. 3.
Critical role of PCNA association and enzyme activities of APE2 in response to hydrogen peroxide-induced damage. (A) Schematic diagram for WT, PIP box mutant (FF), enzyme activity mutants (EA and DA), and CKB mutant (SA) of Myc-APE2. (B) WT or FF 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. (C) Chk1 phosphorylation was analyzed from the samples in B. (D) WT, EA, or DA APE2 was added to APE2-depleted egg extracts supplemented with sperm chromatin and H2O2. Chromatin fractions and total extracts were analyzed for the indicated proteins. (E) Chk1 phosphorylation was examined from the samples in D.
Fig. 4.
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 orthologs 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. 5.
Fig. 5.
A model for APE2 in ATR-Chk1 checkpoint activation in the response to oxidative stress.
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