DNA single-strand breaks (SSBs) can be caused by oxidative stress, or intermediate products of various DNA metabolisms including DNA replication and damage repair. Unrepaired oxidative DNA damage and SSBs may result in replication fork collapse or transcription machinery failure. Oxidative DNA damage and SSBs are critical challenges to genomic stability and can lead to tumorigenesis when they are not repaired quickly or properly. Current understanding of molecular mechanisms underlying checkpoint signaling and regulatory mechanisms in response to oxidative DNA damage and SSBs is limited or indirect because of the lack of feasible experimental systems. Whereas APE1 (AP endonuclease 1) is known for its critical functions in base excision repair and transcriptional regulation, it is currently unknown whether APE1 plays an essential role in DNA damage response (DDR) pathway. Our published work and substantial preliminary data suggest that APE1 is essential for activating the ATR-dependent DDR pathway in oxidative stress, that a distinct ATR-Chk1 checkpoint response is activated by a defined plasmid-based SSB structure, and that APE1 associates with ATRIP and TopBP1. Our major hypothesis is that APE1 plays an vital role in checkpoint signaling in response to oxidative stress and SSBs. To test this directly, our specific aims include: (1) to determine whether APE1 plays an important role in the initiation of SSB end resection in the 3'-5 direction via its exonuclease activity for the SSB signaling; (2) to determine how APE1 interacts with ATRIP in DDR pathway, and (3) to determine how TopBP1 is regulated to activate the ATR-Chk1 checkpoint signaling and whether the role of APE1 in DDR is conserved in pancreatic cancer cells. We have established two complementary approaches to study checkpoint signaling pathway: (1) hydrogen peroxide-induced multiple SSBs randomly distributed on chromatin in a replicating Xenopus LSS system, and (2) plasmid-based site-specific SSB structures in a nonreplicating Xenopus HSS system. Using innovative biochemical and structure-function analysis in Xenopus egg extracts, we will demonstrate how oxidative DNA damage and SSBs are recognized and processed by APE1 in coordination with ATRIP and TopBP1 to regulate checkpoint signaling. We will also validate our findings from Xenopus egg extract system in mammalian cells including pancreatic cancer cells. The anticipated outcomes of this research project will help us better understand how genome stability is maintained in cellular response to oxidative DNA damage and SSBs. All together, this research project will advance our scientific knowledge conceptually on how cancers develop, and open avenues to new therapeutic strategies, especially for pancreatic cancer.
This project uses frog egg extracts and mammalian cell systems to better understand how oxidative stress is sensed and signaled for a checkpoint response. By understanding the mechanisms of oxidative stress signaling, we can better learn how cancer and neurodegenerative disorders develop following oxidative stress, and ultimately treat these diseases via modulating the distinct checkpoint mechanism.