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APE2 promotes DNA damage response pathway from a single-strand break
Yunfeng Lin1, Liping Bai1, Steven Cupello1
1Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA.
Abstract:
As the most common type of DNA damage, DNA single-strand breaks (SSBs) are primarily repaired by the SSB repair mechanism. If not repaired properly or promptly, unrepaired SSBs lead to genome stability and have been implicated in cancer and neurodegenerative diseases. However, it remains unknown how unrepaired SSBs are recognized by DNA damage response (DDR) pathway, largely because of the lack of a feasible experimental system. Here, we demonstrate evidence showing that an ATR-dependent checkpoint signaling is activated by a defined plasmid-based site-specific SSB structure in Xenopus HSS (high-speed supernatant) system. Notably, the distinct SSB signaling requires APE2 and canonical checkpoint proteins, including ATR, ATRIP, TopBP1, Rad9 and Claspin. Importantly, the SSB-induced ATR DDR is essential for SSB repair. We and others show that APE2 interacts with PCNA via its PIP box and preferentially interacts with ssDNA via its C-terminus Zf-GRF domain, a conserved motif found in >100 proteins involved in DNA/RNA metabolism. Here, we identify a novel mode of APE2-PCNA interaction via APE2 Zf-GRF and PCNA C-terminus. Mechanistically, the APE2 Zf-GRF-PCNA interaction facilitates 3'-5' SSB end resection, checkpoint protein complex assembly, and SSB-induced DDR pathway. Together, we propose that APE2 promotes ATR-Chk1 DDR pathway from a single-strand break.
Insights
DNA single-strand breaks (SSBs) trigger ATR-dependent DNA damage response (DDR) signaling. APE2 protein facilitates this response by interacting with PCNA, promoting DNA repair and maintaining genome stability.
Area of Science:
- Molecular Biology
- Cellular Biology
- Genetics
Background:
- DNA single-strand breaks (SSBs) are a common DNA damage type crucial for genome stability.
- Unrepaired SSBs are linked to cancer and neurodegenerative diseases.
- The mechanism of unrepaired SSB recognition by the DNA damage response (DDR) pathway is not well understood.
Purpose of the Study:
- To investigate the mechanism by which unrepaired SSBs are recognized and trigger DDR.
- To elucidate the role of APE2 protein in SSB signaling and repair.
Main Methods:
- Utilized a Xenopus high-speed supernatant (HSS) system with a plasmid-based site-specific SSB structure.
- Investigated the involvement of ATR, ATRIP, TopBP1, Rad9, Claspin, APE2, and PCNA proteins.
- Analyzed protein-protein interactions, including a novel APE2 Zf-GRF and PCNA C-terminus interaction.
Main Results:
- Demonstrated ATR-dependent checkpoint signaling activation by a defined SSB structure.
- Identified APE2 and canonical checkpoint proteins as essential for SSB signaling.
- Showed that APE2-PCNA interaction via APE2 Zf-GRF facilitates DNA end resection, checkpoint assembly, and DDR.
- Confirmed that ATR DDR is essential for SSB repair.
Conclusions:
- APE2 plays a critical role in promoting the ATR-Chk1 DDR pathway activation from SSBs.
- A novel APE2-PCNA interaction mechanism involving APE2's Zf-GRF domain is identified.
- This interaction is crucial for efficient SSB repair and maintaining genome integrity.
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