Mixing active-site components: a recipe for the unique enzymatic activity of a telomere resolvase

Abstract

The ResT protein, a telomere resolvase from Borrelia burgdorferi, processes replication intermediates into linear replicons with hairpin ends by using a catalytic mechanism similar to that for tyrosine recombinases and type IB topoisomerases. We have identified in ResT a hairpin binding region typically found in cut-and-paste transposases. We show that substitution of residues within this region results in a decreased ability of these mutants to catalyze telomere resolution. However, the mutants are capable of resolving heteroduplex DNA substrates designed to allow spontaneous destabilization and prehairpin formation. These findings support the existence of a hairpin binding region in ResT, the only known occurrence outside a transposase. The combination of transposase-like and tyrosine-recombinase-like domains found in ResT indicates the use of a composite active site and helps explain the unique breakage-and-reunion reaction observed with this protein. Comparison of the ResT sequence with other known telomere resolvases suggests that a hairpin binding motif is a common feature in this class of enzyme; the sequence motif also appears in the RAG recombinases. Finally, our data support a mechanism of action whereby ResT induces prehairpin formation before the DNA cleavage step.

Fig. 1.

The hairpin binding motif of cut-and-paste transposases Tn5 and Tn10. (A) The hairpin binding domain bound to Tn5 hairpin DNA. The structural schematic is from the Tn5 crystal structure (Protein Data Bank ID code 1MUS) and was generated by using the vmd molecular graphics program (46). The 5′ phosphate and 3′ hydroxyl group that will take part in forming the interstrand bond are shown in the white and red spheres, respectively. The residue numbering indicated on the structure corresponds to that of the Tn5 transposase amino acid sequence. T2 is the flipped-out thymine base at position 2. (B) Amino acid sequence alignment of ResT (4, 13) with the cut-and-paste transposases of Tn5 and Tn10. Sequence analysis reveals that ResT contains a putative hairpin binding region similar to that found in the Tn5 (27, 28, 32) and Tn10 (31) transposases. The conserved Y-(2)-R-(3)-E-(6)-K signature found in the transposases of IS4 family members (33) is indicated in bold above the alignment. The residues that constitute the hydrophobic binding pocket are colored pink, and those contained in the Y-(2)-R-(3)-E-(6)-K signature are colored blue. Sequence identity is indicated by lighter shades; amino acids with similar properties are denoted by darker shades. The numbering below the alignment corresponds to positions in the ResT sequence.

Fig. 2.

In vitro telomere resolution by ResT. (A Upper) Schematic of the telomere resolution reaction on a double-end-labeled (*), 62-bp, DNA-replicated telomere substrate. Addition of ResT leads to the production of 31-bp resolution products that contain a covalently closed hairpin end. The striped boxes at the ends of the substrate denote different GC clamps, which were required to maximize annealing of the two strands and to minimize snapback of the individual strands on themselves to form hairpins. (A Lower) Autoradiograph of an acrylamide gel showing bands corresponding to the unreacted substrate and the resolution products. A band running at the same position as the product is visible in the unreacted substrate because of some denaturation and subsequent snapback of the very A-T-rich substrate during the 30°C reaction incubation for 30 min. Controls without ResT were run for all substrates, and the background amount of 31-bp hairpin (typically ≈7%) was subtracted from ResT reaction signals. Reactions contained 2 nM labeled substrate and 80 nM ResT and were stopped by the addition of SDS. Products were analyzed on an 8% polyacrylamide gel (see Materials and Methods). (B Upper) Schematic of the telomere resolution reaction on a double-end-labeled (*), 62-bp, asymmetric, DNA-replicated telomere substrate. Addition of ResT leads to the production of 35- and 27-bp resolution products that migrate differently than the unreacted snapback DNA fragment. (B Lower) Autoradiograph of an acrylamide gel showing bands corresponding to the unreacted asymmetric substrate and resolution products.

Fig. 3.

Telomere resolution activity by wild-type and putative hydrophobic binding pocket mutants of ResT. In vitro assays involving resolution of either wild-type or heteroduplex DNA substrates are shown. (A) The symmetric, double-end-labeled, 62-bp wild-type (Top), heteroduplex (Middle), and homoduplex (Bottom) mutant substrates used are shown. The vertical dashed line indicates the axis of symmetry, and the arrows in the wild-type telomere indicate the positions of cleavage. In the heteroduplex DNA substrate, the top strand sequence is wild type throughout, and the bottom strand contains mutations at position 1 flanking the axis of symmetry on both sides. Note that the design of this substrate inhibits base pairing between position-1 nucleotides but allows prehairpinning within each individual strand. The homoduplex mutant substrate contains the same mutation on the bottom strand as in the heteroduplex but also with the complimentary mutation at the top strand to allow base pairing. Mutated bases are shown in bold and boxed in gray. (B) Telomere resolution activity of wild-type ResT, P139A, W141A, and the P139A/W141A double mutant at 30°C. Reactivity levels were determined as the percentage of substrate converted into products. The data shown represent the average of three separate experiments, and the error bars indicate the standard error. (C) DNA cleavage activity of wild-type ResT and ResT with mutations in the putative hairpin binding region. A wild-type substrate carrying 5′ bridging phosphorothiolates at the cleavage positions was used. Cleavage products were detectable as CPDs (see Materials and Methods). The absolute amount of CPD observed with wild-type ResT was 55% of the input substrate. (D) Resolution activity as in B but at 20°C.

Scheme 1.

Scheme 2.

Fig. 4.

Telomere resolution activity by wild-type and YREK mutants of ResT. (A) ResT mutants H324A and Y369F were tested for telomere resolution on symmetric wild-type and heteroduplex substrates to assess specificity of the observed phenotype by hairpin binding mutants of ResT. Reactions at either 30°C (B) or 20°C (C) were performed on YKEK mutants as described in the legend of Fig. 3.

Fig. 5.

Model of the mechanism of the telomere resolution reaction showing the predicted prehairpinning step before the first transesterification. The composite active site is represented by the large gray oval (catalytic residues) and the smaller, darker oval (hairpin binding module). The axis of symmetry is indicated by the dashed line bisecting the telomeric DNA substrate. Prehairpinning is predicted to result in the formation of two small non-base-paired hairpin turnarounds. Although the cis or trans nature of ResT catalysis is not yet determined, the reaction chemistry is depicted in cis by ResT monomers for simplicity.