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.

The translesion DNA synthesis (TLS) polymerase REV1 is implicated in the bypass of the irreparable DNA damage such as interstrand crosslinks (ICLs). However, the potential role of REV1 in DNA damage response (DDR) pathway has not been determined. In this research communication, we provide evidence to demonstrate that REV1 plays a previously unidentified but important role in the ATR-Chk1 checkpoint activation in response to mitomycin C (MMC)-induced ICLs in Xenopus egg extracts. We further pinpointed that REV1 plays a downstream role of a checkpoint protein complex assembly including ATR, ATRIP, TopBP1 and the Rad9-Rad1-Hus1 complex to MMC-induced ICLs on chromatin in the DDR pathway. Notably, domain dissection analysis demonstrates that a C-terminal domain, but not the individual ubiquitin binding motifs, of REV1 is important for the binding of REV1 to MMC-damaged chromatin and the MMC-induced Chk1 phosphorylation. Yet, the ATR-Chk1 DDR pathway appears to be dispensable for the preferential association of REV1 to MMC-damaged chromatin. Taken together, REV1 is important for the DDR pathway in Xenopus egg extracts.

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