PROJECT SUMMARY Since DNA bases are continuously damaged by oxidation, cells have evolved a robust pathway to repair this type of DNA base damage, termed base excision repair (BER). Most oxidative damage can be repaired at the cell’s leisure except at a replication fork, where oxidative damage can cause replication fork collapse. Collapsed forks are a far greater danger to the cell than oxidative damage elsewhere in the genome, but the mechanism of BER at oxidatively damaged replication forks is less well understood compared to BER elsewhere. The 5’ abasic endonuclease APE1 plays a key role in BER repair at oxidatively stressed replication forks. However, there is significant evidence for an alternative pathway; some cancers lack APE1 yet replicate without difficulty, and several aging organs lose expression of APE1 without deleterious effects. We previously found that the 5’ endonuclease EEPD1 can initiate homologous recombination (HR) repair of stressed replication forks by cleaving the lagging parental strand of a stalled fork and loading EXO1 for 5’ end resection in a BRCA1-indepednent manner. In further characterization of EEPD1, we found that it has 5’ abasic endonuclease activity similar but not identical to APE1. EEPD1 can replace APE1 in BER assays in vitro and in vivo. EEPD1 depletion also harmed the repair and restart of oxidatively damaged replication forks. EEPD1 depletion or deletion also resulted in significantly decreased cell survival in the presence of oxidative or alkylative stressors, which cause DNA lesions repaired by BER. EEPD1 has a high differential expression in glioblastoma (GBM) compared to adjacent normal brain or other cancers. GBM exist in a hypoxic environment and are sensitive to oxidative injury, and EEPD1 is required for the survival in every GBM cell line tested. Our SEC-MALS studies found that EEPD1 exists as a dimer in physiologic solution. We resolved the X-ray crystallographic structure of the EEPD1 nuclease domain to 3.0 Å. The tertiary structure of the EEPD1 monomer is similar to the AlphFold2-predicted EEPD1 nuclease domain structure. The EEPD1 crystal structure also has similarities to and distinctions from the APE1 structure. Thus, EEPD1 represents a unique opportunity to gain insight into the structural basis for abasic endonuclease activity and how this activity promotes repair of oxidatively-stressed replication forks. Understanding the structure-function relationship of EEPD1 will lead to regions to target for development of rationally designed inhibitors, for which we have candidate compounds. This is especially important in GBM, for which new therapeutic targets are sorely needed. This renewal application will assess how the structure of EEPD1 functions to repair of replication forks stressed by oxidative DNA damage in GBM cells by addressing three questions: 1) Is EEPD1 dimerization essential for its activity? 2) What EEPD1 domains mediate its 5’ abasic endonuclease activity? 3) What are the dis...