Human FAN1 promotes strand incision in 50-flapped DNA complexed with RPA

yThese authors contributed equally to this work. Fanconi anaemia (FA) is a human infantile recessive disorder. Seventeen FA causal proteins cooperatively function in the DNA interstrand crosslink (ICL) repair pathway. Dual DNA strand incisions around the crosslink are critical steps in ICL repair. FA-associated nuclease 1 (FAN1) is a DNA structure-specific endo-nuclease that is considered to be involved in DNA inci-sion at the stalled replication fork. Replication protein A (RPA) rapidly assembles on the single-stranded DNA region of the stalled fork. However, the effect of RPA on the FAN1-mediated DNA incision has not been deter-mined. In this study, we purified human FAN1, as a bacterially expressed recombinant protein. FAN1 ex-hibited robust endonuclease activity with 50-flapped DNA, which is formed at the stalled replication fork. We found that FAN1 efficiently promoted DNA incision at the proper site of RPA-coated 50-flapped DNA. Therefore, FAN1 possesses the ability to promote the ICL repair of 50-flapped DNA covered by RPA