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...