Abstract 2328 Single Molecule Investigation of Apurinic/Apyrimidinic Endonuclease I (APE1) DNA Damage Search and Recognition

Abasic sites are common mutagenic DNA lesions that occur as products of DNA damage, spontaneously arising up to 10,000 times per cell per day. To prevent mutagenesis, the repair of abasic sites is initiated by the DNA repair enzyme Apurinic/Apyrimidinic Endonuclease I (APE1). While the mechanism used by APE1 to process abasic sites is well established, how APE1 searches for and recognizes abasic sites amongst a vast excess of undamaged DNA remains poorly understood. To address this gap in knowledge, we utilized correlative optical tweezers fluorescence microscopy (CTFM) to visualize the interaction of APE1 with a 12.6 kbp DNA substrate in the presence and absence of a single abasic site. Our initial CTFM analysis on nondamaged DNA identified that APE1 uses a complex mechanism involving both one-dimensional and three-dimensional diffusion during the search for DNA damage. Furthermore, we identified that APE1 can utilize a rapid non-rotationally coupled "hopping" mechanism for one-dimensional diffusion. This combination of one- and three-dimensional diffusion allows APE1 to rapidly sample undamaged DNA for abasic sites across the genome. Additional CTFM analysis also identified that APE1 exhibits longer dwell times on DNA containing an abasic site than on nondamaged DNA, consistent with the ability of APE1 to rapidly recognize abasic sites in the presence of excess undamaged DNA. Together, our characterization of APE1 diffusivity and dwell times indicates that APE1 is a highly efficient DNA repair enzyme that can rapidly identify abasic sites in genomic DNA. This work was funded by the National Institute of Environmental Health Sciences R01ES029203.