An interlocked dimer of the protelomerase TelK distorts DNA structure for the formation of hairpin telomeres

Abstract

The termini of linear chromosomes are protected by specialized DNA structures known as telomeres that also facilitate the complete replication of DNA ends. The simplest type of telomere is a covalently closed DNA hairpin structure found in linear chromosomes of prokaryotes and viruses. Bidirectional replication of a chromosome with hairpin telomeres produces a catenated circular dimer that is subsequently resolved into unit-length chromosomes by a dedicated DNA cleavage-rejoining enzyme known as a hairpin telomere resolvase (protelomerase). Here we report a crystal structure of the protelomerase TelK from Klebsiella oxytoca phage varphiKO2, in complex with the palindromic target DNA. The structure shows the TelK dimer destabilizes base pairing interactions to promote the refolding of cleaved DNA ends into two hairpin ends. We propose that the hairpinning reaction is made effectively irreversible by a unique protein-induced distortion of the DNA substrate that prevents religation of the cleaved DNA substrate.

Figure 1. Protelomerase resolves replicated hairpin telomeres

(A) Replication of a linear chromosome with hairpin telomeres produces a dimeric circular intermediate that is resolved into unit-length chromosomes by the activity of protelomerase. (B) A model for the hairpin formation reaction by the protelomerase TelK, proposed based on the crystal structure presented in this study.

Figure 2. Efficient resolution of the replicated telomere sequence by TelK

Resolution kinetics of 66bp oligonucleotide substrates into the hairpin products by TelK538. A substrate with the natural symmetric central sequence gets resolved efficiently (shown on the gel in the inset), whereas an artificially asymmetrized sequence blocks hairpin formation. Error bars denote standard deviation.

Figure 3. Electron density for the TelK-DNA complex

The simulated annealing composite omit map at 3.2 Å resolution for the TelK-DNA-vanadate complex that represents the intermediate of DNA cleavage reaction. The electron density within 2.5Å from atoms in the final model is shown at a contour level of 1σ above the mean. Densities for protein and DNA are colored in blue and green, respectively. (A) A view perpendicular to the 2-fold noncrystallographic symmetry axis that relates two TelK subunits. (B) Top view along the 2-fold axis.

Figure 4. Structure of the TelK dimer bound to DNA

(A) A view perpendicular to the 2-fold noncrystallographic symmetry axis that relates two TelK subunits colored respectively in orange and cyan. (B) Top view along the 2-fold axis. (C) TelK monomer after a ∼45deg rotation around the vertical axis compared to the cyan monomer in (A), with individual protein domains color-coded. (D) Bottom view along the 2-fold axis showing extensive dimerization contacts between the two catalytic domains.

Figure 5. Distortion of the duplex DNA substrate induced by the binding of a TelK dimer

(A) A large offset in the DNA helical axis is highlighted by dotted lines showing the axes for each half site. (B) Base pairing interactions are severely compromised in the central region between the two scissile phosphates represented by magenta spheres. The unstacked Gua4 bases in turn participate in an inter-strand base stacking. A sigmaA weighted 2Fo-Fc omit map at 3.2Å resolution, contoured at 0.8σ (green) or 1.6σ (red) above the mean level, was computed with phases derived from a model that had never been refined in the presence of the central DNA residues Cyt1, Gua2, Cyt3, Gua4, and Cyt5 built for either strand. (C) The distal region of DNA bound by the stirrup domain is shown for comparison, with a simulated annealing composite omit map (1.0σ) superimposed.

Figure 6. DNA substrate recognition by TelK

(A) A schematic diagram showing the DNA backbone and base-specific interactions. The protein residues are color-coded for each domain according to the scheme in Figure 4C and DNA backbones are colored to match the structural figures. DNA bases involved in hydrogen bonding interactions are colored yellow, and those involved in van der waals contacts are colored cyan. Solid and dashed lines denote electrostatic/hydrogen-bond and van der waals interactions, respectively. (B, C) Base-specific hydrogen bond interactions made by the core-binding and catalytic domains (B), and by the C-terminal stirrup domain (C).

Figure 7. The active site of TelK

(A) Vanadate moiety links 5′- and 3′- ends of DNA to the Tyr425 nucleophile, mimicking the pentavalent DNA cleavage intermediate. (B) Sequences around the catalytically important residues are shown for protelomerase (top), tyrosine recombinase (middle), and type IB topoisomerase (bottom) family of proteins. The aligned proteins are; protelomerases from Klebsiella oxytoca φKO2, Escherichia coli N15, Yersinia enterocolitica PY54, Ectocarpus siliculosus virus, Vibrio harveyi bacteriophage VHML, Agrobacterium tumefaciens, and Borrelia burgdorferi, phage λ integrase, phage P1 Cre, yeast 2μ plasmid Flp, Escherichia coli XerD, phage HP1 integrase, Human topoisomeraseI, Vaccinia virus topoisomerase, Minivirus topoisomerase, and Deinococcus radiodurans topoisomerase IB.