APE1 coordinates its disordered region and metal cofactors to drive genome surveillance.

Efficient recognition of DNA lesions such as apurinic/apyrimidinic (AP) sites is essential for maintaining genome stability. Apurinic/apyrimidinic endonuclease 1 (APE1) is the primary eukaryotic AP endonuclease, yet how it identifies rare lesions among vast stretches of undamaged DNA remains incompletely understood. Using single-molecule imaging combined with molecular dynamics simulations, we reveal that APE1 employs a distinctive dual mechanism to search DNA damage. First, Mg2+ coordination at the active site neutralizes clustered negative charges, stabilizing electrostatic contacts during scanning. Second, its N-terminal intrinsically disordered region (IDR)-a feature conserved only in eukaryotic homologs but absent in prokaryotic ExoIII-not only interacts with DNA through transient IDR contacts but also engages continuous interactions via the unprecedented Arg177 residue within the structured nuclease domain, thereby prolonging residence time and enabling long-range diffusion. Together, these two modules synergize to promote a sliding-based search strategy tailored to the complexity of eukaryotic genomes. Consistent with this model, IDR deletion restricts APE1 to 3D collisions, whereas IDR duplication enhances 1D scanning. Thus, APE1 exemplifies how structural disorder and metal-ion coordination integrate to enable long-range lesion recognition, highlighting an evolutionary innovation in eukaryotic DNA repair.