Abstract 1578 Mechanisms of APE1 in DNA damage response

In our daily lives, cellular DNA is continually subjected to various damaging insults, such as ultraviolet light, chemical agents, reactive oxygen species (ROS), and internal metabolic dysfunctions, leading to the generation of numerous DNA lesions per day. To maintain genomic integrity, organisms activate conserved DNA damage response (DDR) pathways in response to DNA damage and stress conditions. Apurinic/apyrimidinic endonuclease 1 (APE1) exhibits AP endonuclease, 3'-5' exonuclease, 3'-phosphodiesterase, and 3'-exoribonuclease activities and plays central roles in the removal and repair of numerous oxidative DNA lesions including apurinic (AP) sites. However, it remains unclear whether APE1 is involved in DDR pathways. The objective of this research is to elucidate the molecular mechanisms of APE1 in various DDR pathways. Xenopus egg extracts and cultured mammalian cells are utilized as model systems to investigate the mechanisms of APE1 in DDR pathways. We have characterized three distinct mechanisms of APE1 in DDR pathways. Firstly, we have demonstrated that APE1 plays a direct role via its nuclease activity to promote the ATR-Chk1 DDR pathway in response to DNA single-strand breaks in Xenopus egg extracts. Mechanistically, we have found that APE1 can specifically sense and recognize SSB structures and initiate the 3'-5' SSB end resection process through its exonuclease activity, which is continued by APE2 to enlarge the ssDNA for the assembly of ATR/ATRIP complex for activation. Furthermore, APE1 is crucial for ATR-Chk1 DDR pathway activation under stress conditions in human cancer cells, indicating a conserved role of APE1 in ATR-dependent DDR during evolution. Secondly, we have shown that APE1 exhibits a non-nuclease activity role in promoting the nucleolar ATR DDR pathway activation under unperturbed condition in cancer cells. We also have shown that APE1 recombinant protein forms biomolecular condensate via liquid-liquid phase separation in vitro, and that overexpressed APE1 is recruited to nucleoli for nucleolar ATR DDR in dependent of its nuclease activity and redox regulation. The APE1-mediated nucleolar ATR DDR leads to compromised ribosomal RNA transcription and reduced cell viability. Lastly, we have found that APE1 plays a previously uncharacterized but vital role in the DDR pathway mediated by another kinase in the Phosphatidylinositol-3 kinase-related kinases (PIKK) family. In summary, our studies highlight several distinct mechanisms of APE1 in the DDR pathways. Our findings provide novel insights into how genome stability is maintained by APE1. Funding for the research: The Yan lab was supported by the grants from the NIH/NCI (R01CA225637 and R03CA270663) and NIH/NIEHS (R21ES032966), and funds from UNC Charlotte.