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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.

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Figure 1: T5Fen structures and activities.
Figure 2: T5Fen variants D153K and D155K lack enzymatic activity.
Figure 3: Protein-DNA interactions in prethreading T5Fen complex C1.
Figure 4: Structure of a fully threaded DNA complex C2.
Figure 5: Structure of the pseudoenzyme–product complex.
Figure 6: Structural similarities among flap endonucleases.
Figure 7: Overview of the FEN catalytic cycle.

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Acknowledgements

F.A.A. was supported by a scholarship from Taif University. M.F. and C.S.F. were supported by scholarships from the University of Sheffield. Infrastructure was supported through Biotechnology and Biological Research Council (UK) grant awards 50/B19466 (J.R.S.) and REI18458 (J.R.S.).

Author information

Authors and Affiliations

Authors

Contributions

J.R.S., T.C. and P.J.A. conceived the project. J.R.S. designed mutants and enzyme assays. M.F. and J.Z. carried out mutagenesis, biochemical experiments and kinetics analyses (supervised by J.R.S.). F.A.A., C.S.F., M.F. and J.Z. expressed proteins and, together with S.E.S., purified them. F.A.A. carried out crystallization and data collection on complexes (C1–C3). C.S.F. carried out crystallization and data collection on WT T5Fen and the D153K variant. Structure refinement was initially carried out by F.A.A. and C.S.F., under supervision of J.B.R. and P.J.A., and final refinement was performed by J.R.S. with input from J.B.R. and T.C. All authors discussed the results and commented on the manuscript. P.J.A. wrote an initial draft of the manuscript. J.R.S. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to John B Rafferty or Jon R Sayers.

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Competing interests

J.R.S. and J.Z. have filed intellectual property in the area of use of recombinant flap endonucleases (WO 2013079924, World Intellectual Patent Organization). The University of Sheffield has a consultancy agreement with Atlas Genetics Ltd. in the area of flap endonuclease biology (J.R.S.). J.R.S holds equity in a company developing flap endonuclease inhibitors.

Integrated supplementary information

Supplementary Figure 1 Structures of T5Fen and the D153K variant.

(a) Three views of the T5Fen structure showing superposition of the two molecules seen in the asymmetric units (chain A, green; chain B, magenta). (b) Superposition of the helical arch forms of T5Fen (PDB code 5HMM) (grey) with the iso-structural T5FenD153K (PDB code 5HML) molecule (orange). (c) Superposition of looped out forms of wt T5Fen (grey) with the equivalent conformation of D153K molecule (orange). (d) T5Fen sequence with secondary structural elements marked (α-helices as coils; β-strands as arrows). Filled and open circles indicate respectively, direct and indirect ligands for Mg1 (magenta), Mg2 (green) and Mg3 (black). Alpha helical regions are numbered and the position of the H3TH motif is shown. The yellow-boxed region shows residues involved in the alternative conformation for the arch or loop region seen in one chain of each asymmetric unit in the DNA free protein structures.

Supplementary Figure 2 Substrates used for DNA binding and nuclease assays.

(a) Dual-labelled single-turnover substrate OHP2 used for real time endonuclease FRET assay. (b) Diagram of fluorescein-labelled flap substrate used for DNA binding studies. (c) Fluorescence anisotropy was used to determine dissociation constants for the three proteins indicated using the flap substrate shown in (b) in the absence of divalent metal ions (n=3, ±SEM). * P=0.0245, ** P=0.0504 using a 2-tailed t-test.

Supplementary Figure 3 Arrangement of molecules in the asymmetric unit of T5Fen D153K–DNA crystals.

(a) Sequence of oligonucleotide 5ov4, which forms an 8 base-pair palindromic duplex with 4 deoxyadenosines at each 5′ end and was crystalized with T5FenD153K (PDB code 5HNK). (b–d) Three views showing the DNA plus either cartoon (left panels) or molecular surface representations (right panels) of the two protein molecules (chain A in yellow, chain B in grey). Helices 1, 4 and 5 are indicated as well as the 5′ and 3′ ends of DNA strands X (green) and Y (magenta). Two magnesium and potassium ions (orange and purple spheres, respectively) were identifiable in the complex.

Supplementary Figure 4 Arrangement of protein and DNA molecules in the 3′ overhang–T5Fen D155K complex.

(a) Arrangement of three adjacent asymmetric units. Each consists of one protein and one identical DNA (3ov6) molecule (PDB code 5HP4). Two molecules of oligonucleotide (3ov61, orange; 3ov62, blue) form a partially base-paired duplex contacting the central T5Fen molecule (grey surface) while the 3´ end of a third (3ov63, magenta) threads through its helical arch (helices h4 and h5). (b) The central T5Fen protein from (a) showing nucleotides from two adjacent AUs that contact it. The strands have been labeled X, Y and Z. Strand X passed through the helical arch. The resulting assembly resembles a pseudo-product complex such as could have been derived from hydrolysis of the branched substrate at the indicated phosphodiester (inset, red arrow) – or conceptually, by joining strands X and Z to form the cyan strand. (c) The sequence of partially complementary oligonucleotide 3ov6 shown as a duplex. (d) Comparison of position of divalent metal ions in T5Fen (grey cartoon) and the D155K variant (green cartoon). Grey stick residues indicate ligands for Mg2+ ions in M1 and M2 (grey spheres 1 and 2) in the wt protein. In T5FenD155K DNA–Ca2+ complex calcium is positioned at M1 and the ɛ-amino group of Lys155 (blue sphere) is situated close to the M2 site making electrostatic interactions with Asp153 and Asp130. (e) The H3TH motif (a.a. 191–225, grey cartoon) of T5Fen binds a potassium ion (magenta sphere) which in turn binds the phosphate group of dT5 in the duplex DNA. Sequence alignment of the H3TH motifs: residues 191-224 of T5FEN; 163-197 ExoIX and 219-252 from hFEN1 shown below. Consensus sequence shown with similar (:), hydrophilic (%) and not dissimilar (.) residues indicated. (f) Interactions between the 3´ end of one DNA strand with helix 1 (h1). Hydrogen bonds indicated by yellow dashes with water molecules as red spheres. Grey sticks indicate amino acids interacting with DNA.

Supplementary Figure 5 Structural changes in T5Fen after DNA binding.

(a) Schematic showing differences between DNA-bound (complex TC2, magenta) and substrate-free T5Fen structures (green). Residues connecting helices 1 to 2 and 9 to 10 and in helix 4 undergo the largest changes. Helices numbered in white. The junction of helices 4 and 5 is shifted ~ 5-7 Å toward the H3TH motif (h9 and h10) upon binding DNA as shown by the double headed arrow (right panel). (b) Rearrangement of residues 84–92 upon DNA binding. Yellow dashes (with distances) show the largest movements. Atoms of residues labeled in black undergo minimal (<2 Å) translations. Colored labels indicate movement of >2 Å. (c) Two views showing the range of movement observed for helix 4 residues and Arg86 (sticks) in DNA-free, looped-out conformer (cyan cartoon), and with intact substrate bound in complex C2 (magenta cartoon), and in pseudo-product complex C3 (yellow cartoon). The numbered orange spheres show the position of three metal ions (1 and 2 in Cat1 and ion 3 in Cat2. (d) Residues on the helix 1 (h1) and the loop to helix 2 (h2) undergo the next largest rearrangements with His36 playing a role in DNA binding. In one DNA-free form this loop is disordered (not shown). (e) Two views of the H3TH motif (helices h9 and h10) showing residues moving >2 Å upon DNA binding. L202 which changes conformation upon engaging substrate and conserved residues shown in stick representation.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–3 (PDF 2107 kb)

Supplementary Data Set 1

Uncropped gel image for Figure 2a (PDF 19552 kb)

Structure of T5 flap endonuclease and D153K mutant

Wild type structure shown initially, zooms in to show conserved residues and active site with three bound Mg2+ ions (grey sticks) and nearby waters (red spheres). Metal ions 1 and 2 are bound within Cat1, while the third is situated in Cat2. The scene changes to show the the D153K variant (magenta sticks) superposed on the wild type structure. The ɛ-amino group occupies a similar position to that of Mg1 in Cat1 while all other residues remain relatively undisturbed. (MPG 11165 kb)

Model showing DNA bind, thread, bend and barb motions

DNA approaches and binds to the T5Fen protein. A conformational change such as the one shown must occur for DNA to thread through the hole in the enzyme (helical-arch to looped-out form of residues 82–94). The single-stranded end of the DNA then translocates through the protein, which which then undergoes a further conformational change to the helical arch form as a base flips out to pack onto a hydrophobic surface on the distal side of the arch. (MOV 17774 kb)

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AlMalki, F., Flemming, C., Zhang, J. et al. Direct observation of DNA threading in flap endonuclease complexes. Nat Struct Mol Biol 23, 640–646 (2016). https://doi.org/10.1038/nsmb.3241

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