Double strand binding–single strand incision mechanism for human flap endonuclease: Implications for the superfamily

Detailed structural, mutational, and biochemical analyses of human FEN1/DNA complexes have revealed the mechanism for recognition of 5′ flaps formed during lagging strand replication and DNA repair. FEN1 processes 5′ flaps through a previously unknown, but structurally elegant double-stranded (ds) recognition/single stranded (ss) incision mechanism that both selects for 5′ flaps and selects against ss DNA or RNA, intact dsDNA, and 3′ flaps. Two major DNA binding interfaces, including a K+ bridge between the DNA and the H2TH motif, are spaced one helical turn apart and together select for substrates with dsDNA. A conserved helical gateway and a helical cap protects the two-metal active site and selects for ss flaps with free termini. Structures of substrate and product reveal an unusual step between binding substrate and incision that involves a double base unpairing with incision occurring in the resulting unpaired DNA or RNA. Ordering of the active site requires a disorder-to-order transition induced by binding of an unpaired 3′ flap, which ensures that the product is ligatable. Comparison with FEN superfamily members, including XPG, EXO1, and GEN1, identifies superfamily motifs such as the helical gateway that select for ss–dsDNA junctions and provides key biological insights into nuclease specificity and regulation.

► FEN1:DNA structures and mutations reveal 5′-flap recognition mechanism and incision. ► FEN1 recognizes and incises 5′ flaps through dsDNA binding–ssDNA incision. ► FEN1 binds 100° bent dsDNA and unpaired 3′-flap to thread 5′-end near the active site for incision. ► Binding of unpaired 3′ flap and dsDNA induces disorder-to-order transition to align residues for catalysis. ► Two nucleotides of 5′-flap must unpair to productively position phosphodiester near the two catalytic metals.