Direct observation of DNA threading in flap endonuclease complexes

Abstract

Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.9-2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate's single-stranded branch approaches, threads through and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual 'fly-casting, thread, bend and barb' mechanism.

Figure 1

T5Fen structures and activities. (a,b) Cartoon representation of T5Fen in helical arch (a) and looped conformations (b) with helices shown in magenta and β strands in green (numbered in white and black, respectively). (c,d) Close-up view of channel through T5Fen (orange spheres, Cat1 Mg^2+,; black spheres, Mg²⁺ Cat2). (e,f) Side chains of metal-liganding residues shown as sticks, colored according to secondary structure with electron density contoured at 1.2 sigma from a final 2Fo–Fc map (grey mesh) for T5Fen (e) and D153K variant with waters molecules shown as red spheres (f). (g) Cat1 sites of D153K and wt T5Fen superimposed (orange and grey backbones respectively) with grey sphere indicating position of magnesium M1 in (e), and water molecule at M2 site in D153K variant (magenta sphere).

Figure 2

T5Fen variants D153K and D155K lack enzymatic activity. (a) Radiolabelled dT12 (lane 0) reacted with wild type (WT), 153K or 155K variant T5Fen protein as indicated. Lane M, mononucleotide ladder. Original images of the gel can be found in *+. (b) Kinetics of WT T5Fen-catalyzed oligo-dT hydrolysis (error bars represent s.e.m. of 3 technical replicates). (c) Structure-specific endonuclease activity progress curves for WT (+ve), negative control (–ve) and variants indicated. Means (solid lines) and range (dashes) of 3 technical replicates indicated (AU, arbitrary units).

Figure 3

Protein–DNA interactions in pre-threading T5Fen complex C1. (a) Residues within 4 Å of strand X (orange cartoon) or strand Y (green cartoon) colored cyan and magenta, respectively. Helices h4 and h5 indicated. (b) Hydrogen bonds and salt bridges between protein and DNA shown as(orange dashes). Residues within 4 Å shown as sticks. (c) Strand Y nucleotides 8–11 in duplex region (wireframe) contact residues (magenta sticks) in the H3TH motif either via VdW or hydrogen bonds (orange dashes).

Figure 4

Structure of a fully threaded DNA complex C2. (a) Three views of T5FenD153K in complex with Mg²⁺ (blue spheres) and DNA. (b) Contacts to last base pair of duplex (5′dG5:3′dC1 magenta and yellow carbon sticks), showing amino acids within 4 Å (grey sticks). (c) Details of protein interactions with nucleotides dA1–4. (d) Electron density for DNA (magenta mesh, contoured at 1 sigma). Residues within 4 Å on the helical arch in cyan sticks. (e) Stereo diagram showing threaded DNA axis distorted through ~90°. Mg²⁺ ions at M1 and M3 as green spheres. Key residues involved in binding shown as sticks. Waters shown as red spheres, with hydrogen bonds and polar contacts as orange dashes.

Figure 5

Structure of pseudo-enzyme-product complex. (a) Three views of complex. Calcium and potassium ions as blue and magenta spheres, respectively. (b) Electron density contoured at 1.5 sigma for DNA-X (magenta mesh). (c) View corresponding to center panel above. Residues within VdW contact of DNA-X shown as sticks with cyan carbon atoms. Polar contacts (orange dashes) between substrate and bound solvent (red spheres). (d) Detail of active site showing polar contacts around the 3´-end of strand X (magenta) and the 5´-hydroxyl of strand Z (orange). Arg86 contacted strands X and Z. Lys155 occupied the Cat1 M2 site.

Figure 6

Structural similarities between flap endonucleases. (a) Superpositions of T5Fen (magenta cartoons) with E. coli ExoIX (orange cartoon) and human FEN (grey cartoon). (b,c) Views of Cat1 metals (spheres) in T5Fen (magenta sticks) and their ligands superimposed on ExoIX (orange, left panel) and human FEN (grey sticks, right panel).

Figure 7

Overview of FEN catalytic cycle. (a) Substrate binds disordered conformation (top center). Threading occurs (top right) with ordering of h4, barb engagement followed by hydrolysis and product release. (b) Model of active site and scissile nucleotide linkage and water molecule/ OH^– (n, purple sphere) liganded by Mg²⁺ ions (grey spheres). (c) Attack of water molecule n on phosphorus generates a trigonal-bipyramidal transition state. (d) Products bound in active site.