FEN1 participates in repair of the 5′-phosphotyrosyl terminus of DNA single-strand breaks

Etoposide is a widely used anticancer drug and a DNA topoisomerase II (Top2) inhibitor. Etoposide produces Top2-attached single-strand breaks (Top2–SSB complex) and double-strand breaks (Top2–DSB complex) that are thought to induce cell death in tumor cells. The Top2–SSB complex is more abundant than the Top2–DSB complex. Human tyrosyl-DNA phosphodiesterase 2 (TDP2) is required for efficient repair of Top2–DSB complexes. However, the identities of the proteins involved in the repair of Top2–SSB complexes are unknown, although yeast genetic data indicate that 5′ to 3′ structure-specific DNA endonuclease activity is required for alternative repair of Top2 DNA damage. In this study, we purified a flap endonuclease 1 (FEN1) and xeroderma pigmentosum group G protein (XPG) in the 5′ to 3′ structure-specific DNA endonuclease family and synthesized single-strand break DNA substrates containing a 5′-phoshotyrosyl bond, mimicking the Top2–SSB complex. We found that FEN1 and XPG did not remove the 5′-phoshotyrosyl bond-containing DSB substrates but removed the 5′-phoshotyrosyl bond-containing SSB substrates. Under DNA repair conditions, FEN1 efficiently repaired the 5′-phoshotyrosyl bond-containing SSB substrates in the presence of DNA ligase and DNA polymerase. Therefore, FEN1 may play an important role in the repair of Top2–SSB complexes in etoposide-treated cells.