This AREA project will test the hypothesis that APE2 plays an important role in the DNA damage response pathway in response to DNA single-strand breaks (SSBs). SSBs in DNA are a critical challenge to genomic stability and can lead to tumorigenesis and neurodegenerative disorders when they are not repaired quickly or properly. SSBs can be caused by conditions such as oxidative stress, and they can be intermediate products of various DNA metabolisms including DNA replication and DNA repair. SSBs may result in replication fork collapse or transcription machinery failure, or they may become potentially lethal DNA double-strand breaks (DSBs). Current understanding of molecular mechanisms underlying cellular signaling and regulatory mechanisms in response to SSBs is limited or indirect because of the lack of a reliable experiment system. Two complementary approaches have been established to study SSB-induced DNA damage response pathway: (1) hydrogen peroxide-induced indirect SSBs randomly distributed on chromatin in a replicating Xenopus LSS system, and (2) plasmid- based SSB structures at defined locations in a nonreplicating Xenopus HSS system. The PI's published work and substantial preliminary data suggest that a base excision repair protein APE2 plays a vital role in ATR-dependent checkpoint activation in oxidative stress in the LSS, that plasmid-based defined SSBs are resected in the 3'-5' direction, and that a distinct ATR-Chk1 checkpoint response is activated by defined SSB structures. Further mechanistic analysis will provide more details of how the ATR-Chk1 checkpoint is regulated by APE2 in response to SSBs.
Specific aims are: (1) determine the molecular mechanisms of how APE2 senses and binds to DNA SSBs, and (2) determine the distinct mechanisms by which APE2 plays an important role for a defined SSB-induced ATR-Chk1 checkpoint response.
The specific aims i nclude several experiments that will provide multiple research and educational opportunities for students, leading to their publications in peer-reviewed journals and presentations at conferences. The ultimate goal of this project is to better understand how the DNA damage response pathway is activated and regulated in response to DNA SSBs. All together, this research project can advance scientific knowledge conceptually on how cancers or neurodegenerative diseases develop and potentially open up new avenues for therapy. In addition, this project will directly impact research opportunities for the undergraduate and graduate programs and enhance the research environment in the Department of Biological Sciences at the University of North Carolina at Charlotte, enabling more students to be involved in hands-on research projects both independently and in groups.
In order to maintain its genomic integrity and suppress tumors, DNA uses a surveillance mechanism, or DNA damage response pathway, to sense possible damage to its strands. This project uses Xenopus egg extracts as a simple model system to explain how DNA damage, especially single-strand break, is sensed and how DNA damage response pathway is initiated and relayed. Using the details of these essential processes, we will better understand how cancer and neurodegenerative disorders develop, and we can ultimately design innovative processes that manipulate key regulators in DNA damage response to produce novel anti-cancer drugs and therapies.
