Human Flap Endonuclease Structures, DNA Double-Base Flipping, and a Unified Understanding of the FEN1 Superfamily

Abstract

SUMMARY Flap endonuclease (FEN1), essential for DNA replication and repair, removes RNA and DNA 5′-flaps. FEN1 5′ nuclease superfamily members acting in nucleotide excision repair (XPG), mismatch repair (EXO1) and homologous recombination (GEN1) paradoxically incise structurally distinct bubbles, ends or Holliday junctions, respectively. Here structural and functional analyses of human FEN1:DNA complexes show structure-specific, sequence-independent recognition for nicked dsDNA bent 100° with unpaired 3′- and 5′-flaps. Above the active site, a helical cap over a gateway formed by two helices enforces ssDNA threading and specificity for free 5′-ends. Crystallographic analyses of product and substrate complexes reveal that dsDNA binding and bending, the ssDNA gateway, and double-base unpairing flanking the scissile phosphate control precise flap incision by the two-metal-ion active site. Superfamily conserved motifs bind and open dsDNA, direct the target region into the helical gateway permitting only non-base-paired oligonucleotides active site access, and support a unified understanding of superfamily substrate specificity.

The role of DNA exonucleases in protecting genome stability and their impact on ageing

“…Analysis of the structures of FEN1 (Tsutakawa et al 2011) and EXO1 (Orans et al 2011), which were solved at similar times, suggest a unified mechanism for this type of nuclease involving DNA bending and two nucleotide 'fraying' of one strand (Orans et al 2011).…”

“…Furthermore, FEN1 has recently been crystallised both alone (Sakurai et al 2008;Tsutakawa et al 2011) and in association with the Rad9-Rad1-Hus1 (9-1-1) DNA damage checkpoint complex (Dore et al 2009). Analysis of the structures of FEN1 (Tsutakawa et al 2011) and EXO1 (Orans et al 2011), which were solved at similar times, suggest a unified mechanism for this type of nuclease involving DNA bending and two nucleotide 'fraying' of one strand (Orans et al 2011).…”

Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability

“…These 5′ flaps are generated during lagging strand DNA synthesis or during long-patch base excision repair. The FEN1 substrates are in fact double-flap DNA (dfDNA) with DNA on the opposite side of the 5′ flap, forming a single nucleotide 3′ flap when bound to the enzyme (2,3). By removing the 5′ ssDNA or RNA flap from such substrates, FEN1 produces a single nicked product that could be sealed by the subsequent action of a DNA ligase (4).…”

“…Though structural snapshots are available for the individual components of such assemblies (PCNA [Protein Data Bank (PDB) ID code: 1VYM], human 9-1-1 [3GGR]) (Fig. S1) and for two binary complexes [FEN1/DNA (3Q8L) and FEN1/PCNA (1UL1)] (3,16,(18)(19)(20)28), the larger ternary assemblies present Author contributions: J.Q.-A., S.E.T., J.A.T., P.K.C., E.N., and I.I. designed research; J.Q.-A., C.Y., X.X., S.E.T., M.-S.T., and I.I.…”

Okazaki Fragment Metabolism

“…This mechanism was proposed because studies in vitro showed that endonucleolytic cleavage by FEN1 is inhibited when the 5 0 end of the flap is blocked either with a complementary primer or a biotin-conjugated streptavidin moiety (Murante et al 1995). However, recent work has shown that FEN1 initially binds to the base of the flap causing a change in the substrate conformation that orients FEN1 in a manner that allows for precise cleavage (Gloor et al 2010;Tsutakawa et al 2011). The free 5 0 end of the flap is then threaded past or through the helical arch and active site of FEN1 permitting a single cleavage event (Gloor et al 2010).…”