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 AP-sites threaten genomic stability and, if left unrepaired, can lead to mutagenesis and human disease. The essential base excision repair enzyme Apurinic/Apyrimidinic Endonuclease I (APE1) initiates repair by cleaving DNA at AP-sites, yet how APE1 efficiently locates and recognizes these lesions within vast excesses of undamaged DNA has remained poorly understood. Using single-molecule imaging, we show that APE1 employs a dynamic search strategy that integrates 1D and 3D diffusion to rapidly scan DNA. On non-damaged DNA, APE1 undergoes fast 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 into a stationary, lesion-bound complex that is retained at the site of damage. Additional experiments with APE1 variants demonstrate that the intrinsically disordered N-terminal domain of APE1 supports 1D diffusion, whereas residue R177 stabilizes APE1 at the AP-site once recognized, and the catalytic residues D210 and E96 facilitate enzyme release after cleavage. Together, these findings define the molecular basis by which 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 an excess of undamaged DNA. Significance StatementDNA repair enzymes face the formidable challenge of locating rare lesions hidden within vast stretches of undamaged DNA. The essential enzyme APE1 is tasked with identifying and initiating repair of AP-sites, a frequent DNA lesion, to maintain genome integrity. We show that APE1 overcomes this challenge by using a dynamic search mechanism that integrates rapid scanning with precise lesion recognition. Its unstructured N-terminal domain facilitates fast movement along non-damaged DNA, while specific active site residues stabilize engagement at AP-sites and release after catalysis. This coordination of diffusion, recognition, and turnover allows APE1 to efficiently survey the genome and repair AP-sites to maintain genome stability.