Mechanism of APE2 in genome integrity

项目总结/文摘

Project Summary/Abstract Environmental toxins or endogenous insults such as reactive oxygen species (ROS) result in oxidative stress within cells, leading to different types of damage in DNA or RNA, including Apurinic/apyrimidinic (AP) sites and single-strand breaks (SSBs). As the most common type of DNA damage, SSBs have been implicated in association with tumorigenesis, aging, and neurodegenerative disorders. Molecular understanding of SSB repair pathway remains unclear, largely due to the lack of tractable experimental systems. We and others have demonstrated recently that APE2 resolves SSB damage in Xenopus and budding yeast. Although APE1 has been found critical for including DNA repair, redox regulation of transcription, and RNA processing, it remains unclear how APE2 plays important roles in DNA and RNA metabolism. It is significant to determine how APE2 plays an essential role in SSB repair, and how exactly APE2 maintains genome stability. Our substantial preliminary data suggest that APE2’s 3'-5' exonuclease activity is regulated via its dynamic interactions with single-strand DNA (ssDNA) and its interacting proteins, and that APE2 associates with RNA and RNA-containing structures such as R-loop. We will dissect the molecular mechanism of APE2 in metabolism in nucleic acids via two Specific Aims: (1) determine the mechanism of how APE2 interacts with and processes AP sites and SSBs in DNA structures in reconstitution system with purified proteins and in Xenopus egg extracts, and (2) determine how APE2 recognizes and repairs AP site and SSBs in RNA or RNA-containing structures. Biochemical, cell biology, and molecular biology approaches will be utilized to conduct this hypothesis-driven structure-function analysis of APE2-mediated metabolism of nucleic acids in Xenopus egg extracts and reconstitution systems with purified proteins. Anticipated results from this project will make a paradigm shift on APE2- mediated SSB repair in DNA/RNA metabolism to maintain genome stability. Notably, our findings will provide novel insights into new strategies for cancer chemotherapies such as modulating the distinct regulatory mechanisms of APE2 for SSB repair in genome integrity.