Structural basis for the mechanism and stability of the EEPD1 5' endonuclease.

The 5' endonuclease EEPD1 initiates repair of replication forks stalled at oxidative DNA damage. EEPD1 has abasic endonuclease activity that can replace APE1 and initiate base excision repair when the cell is overwhelmed with oxidative DNA damage. In this study, we investigated the structural basis of this activity using X-ray crystallography in conjunction with in vitro endonuclease assays. We resolved the X-ray crystallographic structure of the EEPD1 nuclease domain to 3.2 Å resolution, revealing electrostatic and π-stacking interactions at the homodimeric interface. We further validated the finding that EEPD1 exists as dimers in solution using SEC-MALS analysis, mass photometry, and native gel electrophoresis. Mutations at hydrophobic tryptophans at positions W517, W522, and W524 disrupted the dimerization interface, resulting in a predominantly monomeric EEPD1. While the disruption of dimerization moderately decreased EEPD1's nuclease activity, it significantly decreased its intracellular half-life. We found, as predicted, that catalytic site residues Q269, H404, and D448 are crucial for EEPD1's abasic endonuclease activity, consistent with their structurally predicted role. The EEPD1 catalytic site exhibits geometric conservation of shape and charge in key regions with the APE1's catalytic site, even though these nucleases are otherwise evolutionarily divergent. In summary, these data define the structural basis for the assembly, stability, and endonuclease activity of EEPD1.