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

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 (H 2 O 2 ) 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 H 2 O 2 -induced Chk1 phosphorylation. A conserved proliferating cell nuclear antigen interaction protein box of APE2 is important for the recruitment of APE2 to H 2 O 2 -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. C ells are constantly challenged by exogenous and endogenous insults that threaten genomic integrity. Excess accumulation of reactive oxygen species leads to oxidative DNA damage, such as DNA strand breaks with 3′-modified termini, which is often the underlying pathology in a variety of diseases including neurodegenerative diseases and cancer (1-6). Cellular responses to DNA damage are mainly coordinated by two distinct DNA damage checkpoint signaling cascades: ATM (ataxia-telangiectasia mutated)-checkpoint kinase 2 (Chk2) and ATR (ATM and Rad3-related)-checkpoint kinase 1 (Chk1) pathways (7-10). ATM is activated by intermolecular autophosphorylation and dimer dissociation in response to double-stranded beaks (DSBs) (11-13). ATR is activated by primed single-stranded DNA (ssDNA) in response to a variety of DNA damage or replication stresses (14,15). Oxidative stress has been demonstrated to activate an ATMdependent DNA damage checkpoint (16-18). However, in previous studies, hyperoxic conditions resulted in the phosphorylation of Chk1 and p53 in an ATR-dependent but ATM-independent fashion (19). Furthermore, it remains unclear which specific DNA structures trigger checkpoint signaling during oxidative stress.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, o...

APE2 Zf-GRF facilitates 3′-5′ resection of DNA damage following oxidative stress

“…Histone H3 was used as a loading control for chromatin-bound fractions. These observations suggest that Zf-GRF-stimulated 3′-5′ exonuclease activity of APE2 is important for the generation of RPA-ssDNA; assembly of the checkpoint protein complex including ATR, ATRIP, and 9-1-1 complex; and subsequent Chk1 phosphorylation by activated ATR (22). Similarly, WT APE2, but not the Zf-GRF R502E nuclease-deficient mutant, was able to rescue H 2 O 2 -induced Chk1 phosphorylation in APE2-depleted egg extracts (Fig.…”

“…Disruption of either the APE2 Zf-GRF DNA-binding surface or its PCNA-binding PIP box (22) compromises downstream chromatin recruitment of RPA, checkpoint kinases, and checkpoint activation (SI Appendix , Fig. S13); however, deletion or mutation of the APE2 Zf-GRF has no detectable impact on APE2 association with chromatin following oxidative stress.…”

“…Chk1 phosphorylation serves as an indicator of ATR activation, and activated Chk1 kinase phosphorylates its own substrates, such as Cdc25, to arrest cell cycle progression (20,21). We recently established that the ATR-Chk1 checkpoint is triggered by oxidative DNA damage in Xenopus egg extracts (2,22). Here, checkpoint activation critically requires the APE2 (apurinic/ apyrimidinic endonuclease 2) 3′-5′ resection nuclease.…”

Activation of Checkpoint Kinase Chk1 by Reactive Oxygen Species Resulting from Disruption of wat1/pop3 in Schizosaccharomyces pombe

“…The phosphorylation of Chk1 and increased Rad22-YFP foci in wat1-null mutants, even in the absence of genotoxic stimuli, support our hypothesis. Since phosphorylation of Chk1 in response to H 2 O 2 has been reported recently (Willis et al 2013), and Wat1 is a major component of both the Tor2 and Tor1 complexes (TORC1 and TORC2), we speculated that it plays a role in the regulation of the oxidative stress response. We observed much reduced growth of wat1-deleted cells on plates containing H 2 O 2, indicating its role in the oxidative stress response.…”

ATR maintains chromosomal integrity during postnatal cerebellar neurogenesis and is required for medulloblastoma formation

“…ATR is activated in response to stalled DNA replication forks (Nam and Cortez, 2011), which can be caused by exogenous (Harley et al, 2016;Harper et al, 2010) or endogenous (Willis et al, 2013) factors. We have previously shown that normal CGNP proliferation produces endogenous DNA damage, detectable as small γH2A.X + foci (Williams et al, 2015).…”