Significance Zf-GRF domains are found in more than 100 eukaryotic architectures, including key proteins modulating DNA damage response and transcription. We establish the apurinic/apyrimidinic endonuclease 2 (APE2) Zf-GRF domain as a prototypical member of the Zf-GRF class of nucleic acid-binding modules, and through structural analysis reveal that the APE2 protein is composed of a compacted three-stranded β-sheet and a CHCC Zn ²⁺ -binding site, harboring structure-specific ssDNA-binding activity. Notably, the ssDNA-binding region of APE2 Zf-GRF is required for the 3′-5′ end resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response pathway following oxidative stress. This distinct regulatory mechanism of APE2 exonuclease activity by ssDNA binding via Zf-GRF may extend to other Zf-GRF–containing proteins.