Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily

Abstract

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 nonbase-paired oligonucleotides active site access; and support a unified understanding of superfamily substrate specificity.

Figure 1. Binding of 5′-Flap DNA in FEN1:Sm³⁺:Product DNA Complex

(All panels) FEN1-bound DNA includes 3′-flap strand (magenta), 5′-flap strand (purple), and template strand (brown). (A) The 4 nt flap crystallization substrate numbered relative to scissile phosphate. The flap strand (+1 to +4 nts) is absent in the WT:Sm³⁺: product complex. The 1 nt flap substrate lacks the +2 to +4 nts. (B) FEN1 surface and F_o-F_c electron density of DNA from PHENIX kick map shows clear density for 100° bent DNA bound by FEN1 (1.6σ map within 1.9 Å of DNA). Crystallographic statistics are in Table S1. FEN1 preferentially interacts with the template strand and the 3′- and 5′ -flap terminal nucleotides (Figure S1A) (C) FEN1:DNA showing the structural elements involved in DNA binding and the bound DNA distortion. Key elements include the hydrophobic wedge that breaks DNA path and forms the 3′-flap binding site (green), acid block that inhibits longer 3′-flaps (red), helical gateway that permits only ssDNA and forms the active site (dark blue), helical cap that imposes the preference for 5′-termini (pink), and H2TH that binds to dsDNA upstream from 5′-flap (purple). Sm³⁺ (cyan) and K⁺ (purple) ions are shown as spheres. Comparison with FEN1:substrate complexes is shown in Figures S1B-D. (D) Electrostatic surface (-52 mV to +52 mV) of FEN1 and cations shows how the template strand traverses the path of basic residues. (E) Key structural elements in FEN1 involved in DNA recognition and incision. (F) Schematic of all direct protein-DNA interactions shows that binding is concentrated to template strand and are not base-specific. Numbering is based on the pdb. (G) A transparent FEN1 surface reveals binding sites to template dsDNA strands are spaced ~1 helical turn apart. Fixed by the bending point of DNA and the 3′ -flap binding, binding of the template strand positions the complementary strand (the 5′ -flap) at the gateway under the cap.

Figure 2. Sequence, Secondary Structure and Residue Function for FEN1 and the 5′ Nuclease Superfamily

Map of FEN1 secondary structure, structural elements, and mutants to a sequence alignment of the FEN1 superfamily human members. XPG residues 121-750 were removed (dotted line) to facilitate alignment.

Figure 3. Key FEN1 Structural Elements in the FEN:Sm³⁺:Product Complex

(A) The H2TH:K⁺ motif and surrounding basic residues (purple) forms an electrostatic track for the downstream dsDNA minor groove. (B) The 3′-flap binding pocket (green) binds the 3′-hydroxyl and the unpaired nt sugar moiety. (C) Rear view of the active site, opposite the template DNA interacting region, shows the helical gateway (blue), active site residues and product DNA. The cleaved 5′-nt sits in the helical gateway formed by α2 and α4 and under the cap. Figures S2A-C show C-capping in the gateway. (D) Front view of the active site shows the gateway and 5′-flap strand approach to the active site. (E) FEN1 binds to the -1 to -3 phosphates of the 5′ -flap product strand. (F) Four conserved FEN1 carboxylate residues and two phosphate oxygens of the +1 5′-phosphate in the DNA directly coordinate the two Sm³⁺ ions. Distances are 2.3 – 2.5 Å. Figure S2D shows all seven highly conserved carboxylates and bound waters.

Figure 4. Substrate and Product Complex DNA Structure Comparison Reveals a Novel Double Base Unpairing Mechanism for Scissile Phosphate Placement

(A) DNA from WT:substrate and product complexes show the +1 and -1 nts are paired in the substrate but the -1 nt in the product is unpaired with Sm³⁺ ions (green spheres, panels A, C-E). (B) Close-up of the DNA and Tyr40 from the D181A:substrate complex showing the basepairing of the +1 and -1 nts and Tyr40 stacking with the +1 nt. The overlay with the product structure outline highlights the 7.7 Å movement of the scissile phosphate of the -1 nt (green) needed for catalysis. (C) As in (B), the +1 and -1 nt are basepaired and Tyr40 stacks with the +1 nt in the Wt:Sm³⁺:substrate. The scissile phosphate of the -1 nt would need to move 5.2 Å into the active site. (D) Close-up view of the DNA and Tyr40 from the WT:Sm³⁺:product DNA complex. Unlike the substrate complexes, the +1 nt has been cleaved off, the -1 nt is unpaired, and Tyr40 stacks with the -1 nt. (E) Model of double nt unpairing to move substrate into a catalytic position for incision.

Figure 5. Structure–Guided Site-Directed Mutagenesis of Active Site, Helical Gateway, Hydrophobic Wedge and Helical Cap Residues

(A) FEN1:Sm³⁺:product complex fold (ribbons) marking the position of mutations in this study (colored as in Figure 1). (B) Substrate used in (C) with schematic of the incision. (C) Catalytic efficiency of WT and mutant FEN1 as bar graph showing the relative severity of mutations. Controls and examples of data are in Figure S3. (D) 5′ -phosphate substrates used in (E) with schematic of the incision. (E) Denaturing PAGE gel showing increased incision activity on substrates with a 5′-phosphate at the +1 position.

Figure 6. FEN1 Implications for XPG

(A) Denaturing PAGE gel showing reduced bubble incision activity by XFX2. XPG, XFX2, and FEN1 incision activity of 30 nt DNA bubbles at 1 nM protein concentration. Incubation time was 5, 15, 30, 60, 120 min. Substrate sequences are in Figure S4. (B) Denaturing PAGE gel showing XFX2 is active on 5′-flap DNA. FEN1 and XFX2 were incubated for 15, 30, and 60 min. (C) Quantification of (A) (D) Quantification of (B)

Figure 7. FEN1 Primarily Binds and Bends dsDNA to Detect dsDNA Junctions and Undergoes Disorder-To-Order Transitions to Recognize 5′-flaps

(A) FEN1 binds primarily to downstream dsDNA by the K⁺:H2TH, allowing FEN1 to search for target DNA structure. The second juxtaposed binding site selects for dsDNA structures that can sharply bend ~100°. Coincident with DNA template strand binding, the 5′-ss flap is directed under the disordered cap domain and through the helical gateway selecting for ssDNA nearing the active site. Assembly of the 3′-flap site, cap ordering and double base unpairing promote correct positioning of the scissile bond and rapid two-metal-ion catalyzed incision. Movie S1 shows morphing between models of three DNA-free FEN1 structures and the product DNA-bound structure. (B) Overlay of DNA from complexes with FEN1 (product) and Pol β (1TV9) (Krahn et al., 2004) reveals that Pol β binding does not block the FEN1 downstream dsDNA binding region, suggesting a baton passing mechanism for direct handoff. (Left) DNA atoms shown within 4 Å of protein in the complex (FEN1, purple; Pol β, blue, colored spheres). (Right) Pol β shown with DNA overlay. (C) Both FEN1 and the Ligase I DBD can bind simultaneously to the DNA, based on an overlay of 5′ -flap DNA from complexes with FEN1 (product) and Ligase I DBD (1X9N) (Pascal et al., 2004). (Left) DNA atoms shown within 4 Å of protein in the complex (FEN1, purple; Ligase I DBD, green, colored spheres). (Right) Overlaid complexes.