A dynamic search mechanism enables APE1 to identify AP-sites in DNA.

Apurinic/apyrimidinic (AP) sites are among the most frequent DNA lesions, arising thousands of times per cell each day. These lesions threaten genomic stability and, if left unrepaired, can lead to mutagenesis and disease. The base excision repair enzyme Apurinic/Apyrimidinic Endonuclease I (APE1) initiates repair by cleaving DNA at AP-sites, yet how APE1 efficiently identifies these lesions within vast excesses of undamaged DNA has remained unclear. Using single-molecule imaging, we show that APE1 employs a dynamic search strategy that integrates one-dimensional (1D) and three-dimensional (3D) diffusion to rapidly scan DNA. On non-damaged DNA, APE1 undergoes rapid diffusion, enabling efficient interrogation of large genomic regions within a single binding event. Upon encountering an AP-site, APE1 transitions from a mobile search state to a stable lesion-bound complex that remains localized at the site of damage. Experiments with APE1 variants further reveal that the intrinsically disordered N-terminal domain promotes 1D diffusion, residue R177 stabilizes APE1 at the AP-site after recognition, and catalytic residues D210 and E96 facilitate enzyme release following cleavage. Together, these findings define how APE1 balances rapid genome surveillance with stable lesion engagement and timely release. More broadly, this work provides a mechanistic framework for how DNA repair enzymes efficiently locate and process rare lesions embedded within excess undamaged DNA.