Preventing broken Borrelia telomeres: ResT couples dual hairpin telomere formation with product release

Spirochetes of the genus Borrelia include the tick-transmitted causative agents of Lyme disease and relapsing fever. They possess unusual genomes composed mainly of linear replicons terminated by closed DNA hairpins. Hairpin telomeres are formed from inverted repeat replicated telomere junctions (rTels) by the telomere resolvase ResT. ResT uses a reaction mechanism similar to that of the type IB topoisomerases and tyrosine recombinases. ResT can catalyze three distinct reactions: telomere resolution, telomere fusion, and Holliday junction (HJ) formation. HJ formation is known to occur only in the context of a synapsed pair of rTels. To test whether telomere resolution was synapsis-dependent, we performed experiments with rTel substrates immobilized on streptavidin-coated beads. We report that telomere resolution by ResT is synapsis-independent, indicating that alternative complexes are formed for telomere resolution and HJ formation. We also present evidence that dual hairpin telomere formation precedes product release. This mechanism of telomere resolution prevents the appearance of broken telomeres. We compare and contrast this mechanism with that proposed for TelK, the telomere resolvase of φKO2.     

Fusion of hairpin telomeres by the B. burgdorferi telomere resolvase ResT implications for shaping a genome in flux

Spirochetes of the genus Borrelia include the causative agents of Lyme disease and relapsing fever. These bacteria have a highly segmented genome where most replicons are linear molecules terminated by covalently closed hairpin telomeres. Moreover, these genomes appear to be in a state of flux with extensive and ongoing DNA rearrangements by unknown mechanisms. The B. burgdorferi telomere resolvase ResT generates the hairpin telomeres from replication intermediates in a reaction with mechanistic similarities to that catalyzed by type IB topoisomerases and tyrosine recombinases. We report here the unexpected ability of ResT to catalyze the fusion of hairpin telomeres in a reversal of the telomere resolution reaction. We propose that stabilized ResT-mediated telomere fusions are an underlying force for maintaining the B. burgdorferi genome in a state of flux.     

Construction and Characterization of a Borrelia burgdorferi Strain with Conditional Expression of the Essential Telomere Resolvase,ResT

Borrelia species are unique in the bacterial world in possessing segmented genomes which sometimes contain over 20 genetic elements. Most elements are linear and contain covalently closed hairpin ends requiring a specialized process, telomere resolution, for their generation. Hairpin telomere resolution is mediated by the telomere resolvase, ResT. Although the process has been studied extensively in vitro, the essential nature of the resT gene has precluded biological studies to further probe the role of ResT. In this work, we have generated a B. burgdorferi strain that carries an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible resT gene controlled by a tightly regulated promoter. ResT is expressed in this strain at ∼14,000 monomers per cell, similar to the ∼15,000 monomers observed for the parental strain. We demonstrate ResT depletion with a half-life of 16 h upon IPTG washout. ResT depletion resulted in arrested growth 48 h after washout. Interestingly, not all spirochetes died after ResT washout, and at least 15% remained quiescent and could be resuscitated even at 2 weeks postwashout. Significant levels of DNA synthesis were not observed upon growth arrest, suggesting that ResT might interact directly or indirectly with factors controlling the initiation or elongation of DNA synthesis. Analysis of the linear plasmids lp17 and lp28-2 showed that the linear forms of these plasmids began to disappear and be replaced by higher-molecular-weight forms by 24 h post-IPTG washout. Treatment of DNA from the ResT-depleted strain with ResT in vitro revealed the presence of replicated telomeres expected in replication intermediates.     

Hairpin telomeres and genome plasticity in Borrelia: all mixed up in the end

Spirochetes of the genus Borrelia have a highly unusual genome structure composed of over 20 replicons. Most of these replicons are linear and terminated by covalently closed hairpin ends or telomeres. Moreover, the linear replicons are affected by extensive DNA rearrangements, including telomere exchanges, DNA duplications, and harbour a large number of pseudogenes. The mechanism for the unusual genome plasticity in the linear replicons has remained elusive. The enzymatic machinery (the telomere resolvase ResT) responsible for generating the hairpin ends from replicative intermediates has recently been shown to also perform a reverse reaction that fuses telomeres on unrelated replicons. Infrequent stabilization of such fusion events over evolutionary time provides the first proposed biochemical mechanism for the DNA rearrangements that are so prominent in the linear replicons of B. burgdorferi.     

Holliday junction formation by the Borrelia burgdorferi telomere resolvase, ResT: implications for the origin of genome linearity

Spirochetes of the genus Borrelia include the causative agents of Lyme disease and relapsing fever. They possess unusual, highly segmented genomes composed mostly of linear replicons with covalently closed hairpin telomeres. The telomeres are formed from inverted repeat replicated telomere junctions ( rTel s) by the telomere resolvase, ResT. ResT uses a reaction mechanism with similarities to that employed by the type IB topoisomerases and tyrosine recombinases. Here, we report that the relationship of ResT to the tyrosine recombinases extends to the ability to synapse-replicated telomeres and to catalyse the formation of a Holliday junction. We also report that ResT can use asymmetrized substrates that mimic the properties of a recombination site for a tyrosine recombinase, to form Holliday junctions. We propose a model for how this explains the origin of genome linearity in the genus Borrelia.     

Uncoupling the chemical steps of telomere resolution by ResT

ResT is the telomere resolvase of the spirochete Borrelia burgdorferi, the causative agent of Lyme disease. ResT is an essential cellular function that processes replication intermediates to produce linear replicons terminated by covalently closed hairpin telomeres. ResT generates these hairpin telomeres in a reaction with mechanistic similarities to those catalyzed by type IB topoisomerases and tyrosine recombinases. We report here, that like most of the tyrosine recombinases, ResT requires interprotomer communication, likely in an in-line synapse, to activate reaction chemistry. Unlike the tyrosine recombinases, however, we infer that the cleavage and strand transfer reactions on the two sides of the replicated telomere occur nearly simultaneously. Nonetheless, the chemical steps of the forward and reverse reactions performed by ResT can occur in a non-concerted fashion (i.e. events on the two sides of the replicated telomere can occur independently). We propose that uncoupling of reaction completion on the two sides of the substrate is facilitated by an early commitment to hairpin formation that is imposed by the precleavage action of the hairpin binding module of the ResT active site.     

Telomere resolution by Borrelia burgdorferi ResT through the collaborative efforts of tethered DNA binding domains

Borrelia burgdorferi, a causative agent of Lyme disease, has a highly unusual segmented genome composed of both circular molecules and linear DNA replicons terminated by covalently closed hairpin ends or telomeres. Replication intermediates of the linear molecules are processed into hairpin telomeres via the activity of ResT, a telomere resolvase. We report here the results of limited proteolysis and mass spectroscopy to identify two main structural domains in ResT, separated by a chymotrypsin cleavage site between residues 163 and 164 of the 449 amino acid protein. The two domains have been overexpressed and purified. DNA electrophoretic mobility shift assays revealed that the C-terminal domain (ResT(164-449)) displays sequence-specific DNA binding to the box 3,4,5 region of the telomere, while the N-terminal domain (ResT(1-163)) exhibits sequence-independent DNA binding activity. Further analysis by DNase I footprinting supports a model for telomere resolution in which the hairpin binding module of the N-terminal domain is delivered to the box 1,2 region of the telomere through its tethering to ResT(164-449). Conversely, ResT(1-164) may play an important regulatory role by modulating both sequence-specific DNA binding activity and catalysis by the C-terminal domain.     

Spring loading a pre-cleavage intermediate for hairpin telomere formation

The Borrelia telomere resolvase, ResT, forms the unusual hairpin telomeres of the linear Borrelia replicons in a process referred to as telomere resolution. Telomere resolution is a DNA cleavage and rejoining reaction that proceeds from a replicated telomere intermediate in a reaction with mechanistic similarities to that catalyzed by type IB topoisomerases. Previous reports have implicated the hairpin-binding module, at the end of the N-terminal domain of ResT, in distorting the DNA between the scissile phosphates so as to promote DNA cleavage and hairpin formation by the catalytic domain. We report that unwinding the DNA between the scissile phosphates, prior to DNA cleavage, is a key cold-sensitive step in telomere resolution. Through the analysis of ResT mutants, rescued by substrate modifications that mimic DNA unwinding between the cleavage sites, we show that formation and/or stabilization of an underwound pre-cleavage intermediate depends upon cooperation of the hairpin-binding module and catalytic domain. The phenotype of the mutants argues that the pre-cleavage intermediate promotes strand ejection to favor the forward reaction and that subsequent hairpin capture is a reversible reaction step. These reaction features are proposed to promote hairpin formation over strand resealing while allowing reversal back to substrate of aborted reactions.     

Split target specificity of ResT: a design for protein delivery,site selectivity and regulation of enzyme activity?

The ResT telomere resolvase is responsible for maintaining the hairpin telomeres that cap the linear chromosome and minichromosomes of Borrelia burgdorferi. This enzyme acts at the tandem telomere junctions present within circular dimers resulting from DNA replication. ResT mediates the transesterification steps of resolution using a constellation of active site residues similar to that found in tyrosine recombinases and type IB topoisomerases. By combining this reaction mechanism with a hairpin binding module in its N-terminal domain, ResT reduces a fused telomere dimer into two hairpin monomers. ResT displays a split DNA binding specificity, with the N- and C-terminal domains targeting distinct regions of the telomere. This bi-specificity in binding is likely to be important in protein delivery, substrate selection and regulation of enzyme activity.     

Bidirectional replication from an internal ori site of the linear N15 plasmid prophage

The prophage of coliphage N15 is not integrated into the chromosome but exists as a linear plasmid molecule with covalently closed hairpin ends (telomeres). Upon infection the injected phage DNA circularizes via its cohesive ends. Then, a phage-encoded enzyme, protelomerase, cuts the circle and forms the hairpin telomeres. N15 protelomerase acts as a telomere-resolving enzyme during prophage DNA replication. We characterized the N15 replicon and found that replication of circular N15 miniplasmids requires only the repA gene, which encodes a multidomain protein homologous to replication proteins of bacterial plasmids replicated by a theta-mechanism. Replication of a linear N15 miniplasmid also requires the protelomerase gene and telomere regions. N15 prophage replication is initiated at an internal ori site located within repA and proceeds bidirectionally. Electron microscopy data suggest that after duplication of the left telomere, protelomerase cuts this site generating Y-shaped molecules. Full replication of the molecule and subsequent resolution of the right telomere then results in two linear plasmid molecules. N15 prophage replication thus appears to follow a mechanism that is distinct from that employed by eukaryotic replicons with this type of telomere and suggests the possibility of evolutionarily independent appearances of prokaryotic and eukaryotic replicons with covalently closed telomeres.     

Sequence-specific recognition but position-dependent cleavage of two distinct telomeres by the Borrelia burgdorferi telomere resolvase,ResT

An unusual feature of bacteria in the genus Borrelia (causative agents of Lyme disease and relapsing fever) is a segmented genome consisting of multiple linear DNA molecules with covalently closed hairpin ends, known as telomeres. The hairpin telomeres are generated by a DNA breakage and reunion process (telomere resolution) promoted by ResT, an enzyme using an active site related to that of tyrosine recombinases and type IB topoisomerases. In this study, we define the minimal sequence requirements for a functional telomere and identify specific basepairs that appear to be important for telomere resolution. In addition, we show that the two naturally occurring and distinct telomere spacings found in B. burgdorferi can both be efficiently processed by ResT. This flexibility for substrate utilization by ResT supports the argument for a single telomere resolvase in Borrelia. Furthermore, although telomere recognition requires sequence specificity in part of the substrate, DNA cleavage is instead position dependent and occurs at a fixed distance from the axis of symmetry and the conserved sequence of box 3 in the different replicated telomere substrates. This positional dependence for DNA cleavage has not been observed previously for a tyrosine recombinase.     

Telomere resolution in the Lyme disease spirochete.

The genus Borrelia includes the causative agents of Lyme disease and relapsing fever. An unusual feature of these bacteria is a genome that includes linear DNA molecules with covalently closed hairpin ends referred to as telomeres. We have investigated the mechanism by which the hairpin telomeres are processed during replication. A synthetic 140 bp sequence having the predicted structure of a replicated telomere was shown to function as a viable substrate for telomere resolution in vivo, and was sufficient to convert a circular replicon to a linear form. Our results suggest that the final step in the replication of linear Borrelia replicons is a site-specific DNA breakage and reunion event to regenerate covalently closed hairpin ends. The telomere substrate described here will be valuable both for in vivo manipulation of linear DNA in Borrelia and for in vitro studies to identify and characterize the telomere resolvase.     

ResT, a telomere resolvase encoded by the Lyme disease spirochete.

The genus Borrelia includes the causative agents of Lyme disease and relapsing fever. An unusual feature of these bacteria is a segmented genome consisting mostly of a number of linear DNA molecules with covalently closed hairpin ends or telomeres. In this study we show that the BBB03 locus encodes the B. burgdorferi telomere resolvase, ResT. The purified protein catalyzes telomere resolution in vitro through a unique reaction: breakage of two phosphodiester bonds in a single DNA duplex (one on each strand) and joining of each end with the opposite DNA strand to form covalently closed hairpin telomeres. Telomere resolution by ResT occurs through a two-step transesterification reaction involving the formation of a covalent protein-DNA intermediate at a position three nucleotides from the axis of symmetry in each strand of the substrate.     

Differential telomere processing by Borrelia telomere resolvases in vitro but not in vivo

Causative agents of Lyme disease and relapsing fever, including Borrelia burgdorferi and Borrelia hermsii, respectively, are unusual among bacteria in that they possess a segmented genome with linear DNA molecules terminated by hairpin ends, known as telomeres. During replication, these telomeres are processed by the essential telomere resolvase, ResT, in a unique biochemical reaction known as telomere resolution. In this study, we report the identification of the B. hermsii resT gene through cross-species hybridization. Sequence comparison of the B. hermsii protein with the B. burgdorferi orthologue revealed 67% identity, including all the regions currently known to be crucial for telomere resolution. In vitro studies, however, indicated that B. hermsii ResT was unable to process a replicated B. burgdorferi type 2 telomere substrate. In contrast, in vivo cross-species complementation in which the native resT gene of B. burgdorferi was replaced with B. hermsii resT had no discernible effect, even though B. burgdorferi strain B31 carries at least two type 2 telomere ends. The B. burgdorferi ResT protein was also able to process two telomere spacing mutants in vivo that were unresolvable in vitro. The unexpected differential telomere processing in vivo versus in vitro by the two telomere resolvases suggests the presence of one or more accessory factors in vivo that are normally involved in the reaction. Our current results are also expected to facilitate further studies into ResT structure and function, including possible interaction with other Borrelia proteins.     

Replication at the telomeres of the Streptomyces linear plasmid pSLA2

The Streptomyces linear plasmid pSLA2 initiates DNA replication bidirectionally towards its telomeres from a site located near the centre of the molecule; at the telomeres, the recessed ends of lagging strands are filled in by non-displacing DNA synthesis. Here, we report experiments that test three proposed mechanisms for lagging-strand fill-in. We present data inconsistent with recombinational or terminal hairpin models for the formation of full-length duplex pSLA2 DNA. Instead, we find that deletions in short, distantly separated homologous palindromes in the leading-strand 3' overhang prevent propagation of linear pSLA2 DNA, implicating a mechanism of palindrome-mediated leading-strand fold-back in telomere replication. We further show that circularized pSLA2 DNA molecules are opened in vivo precisely at the terminal nucleotides of telomeres, generating functional linear replicons containing native telomeres covalently bound to a protein at their 5' DNA termini. Together, our results support a model in which pairing of multiple widely separated pSLA2 palindromes anchors the 3' end of the leading-strand overhang to a site near the overhang's base — providing a recognition site for terminal-protein-primed DNA synthesis and subsequent endonucleolytic processing. Thus, the replication of Streptomyces plasmid telomeres may have features in common with the mechanism proposed for telomere replication in autonomous parvoviruses.     

Survival mechanisms for Streptomyces linear replicons after telomere damage

The ability of linear replicons to propagate their DNA after telomere damage is essential for perpetuation of the genetic information they carry. We introduced deletions at specific locations within telomeres of streptomycete linear plasmids and investigated mechanisms that enable survival. Here, we report that rescue of such plasmids in Streptomyces lividans occurs by three distinct types of events: (i) repair of the damaged telomere by homologous recombination; (ii) circularization of the plasmid by non-homologous end-to-end joining; and (iii) formation of long palindromic linear plasmids that duplicate the intact telomere by a non-recombinational process. The relative frequency of use of these survival mechanisms depended on the location and length of the telomeric DNA deletion. Repair by intermolecular recombination between the telomeres of chromosomes and plasmids, deletion of additional DNA during plasmid circularization, and insertion of chromosomal DNA fragments into plasmids during end-to-end joining were observed. Our results show that damage to telomeres of Streptomyces linear replicons can promote major structural transformations in these replicons as well as genetic exchange between chromosomes and extrachromosomal DNA. Our findings also suggest that spontaneous circularization of linear Streptomyces chromosomes may be a biological response to instances of telomere damage that cannot be repaired by homologous recombination.     

An Enzyme-Catalyzed Multistep DNA Refolding Mechanism in Hairpin Telomere Formation

Hairpin telomeres of bacterial linear chromosomes are generated by a DNA cutting–rejoining enzyme protelomerase. Protelomerase resolves a concatenated dimer of chromosomes as the last step of chromosome replication, converting a palindromic DNA sequence at the junctions between chromosomes into covalently closed hairpins. The mechanism by which protelomerase transforms a duplex DNA substrate into the hairpin telomeres remains largely unknown. We report here a series of crystal structures of the protelomerase TelA bound to DNA that represent distinct stages along the reaction pathway. The structures suggest that TelA converts a linear duplex substrate into hairpin turns via a transient strand-refolding intermediate that involves DNA-base flipping and wobble base-pairs. The extremely compact di-nucleotide hairpin structure of the product is fully stabilized by TelA prior to strand ligation, which drives the reaction to completion. The enzyme-catalyzed, multistep strand refolding is a novel mechanism in DNA rearrangement reactions.     

The protelomerase of the phage-plasmid N15 is responsible for its maintenance in linear form

The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularises via cohesive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). We demonstrate that this enzyme acts in vivo on specific substrates, and show that it is necessary for replication of the linear prophage. We show that protelomerase is an end-resolving enzyme responsible for processing of replicative intermediates. Removal of protelomerase activity resulted in accumulation of replicative intermediates that were found to be circular head-to-head dimers. N15 protelomerase and its target site constitute a functional unit acting on other replicons independently of other phage genes; a mini-F or mini-P1 plasmid carrying this unit replicates as a linear plasmid with covalently closed ends. Our results suggest the following model of N15 prophage DNA replication. Replication is initiated at an internal ori site located close to the left end of plasmid DNA and proceeds bidirectionally. After replication of the left telomere, protelomerase cuts this sequence and forms two hairpin loops telL. After duplication of the right telomere (telR) the same enzyme resolves this sequence producing two linear plasmids. Alternatively, full replication of the linear prophage to form a circular head-to-head dimer may precede protelomerase-mediated formation of hairpin ends.     

Replication timing of large <Emphasis Type="Italic">Sorex granarius</Emphasis> (<Emphasis Type="Italic">Soricidae</Emphasis>, <Emphasis Type="Italic">Eulipotyphla</Emphasis>) telomeres

Previously, we described the unique feature of telomeric regions in Iberian shrew Sorex granarius: its telomeres have two ranges of size, very small (3.8 kb of telomeric repeats on average) and very large discontinuous telomeres (213 kb) interrupted with 18S rDNA. In this study, we have demonstrated extraordinary replication pattern of S. granarius large telomeres that have not been shown before in other studied mammal. Using the ReD-FISH procedure, we observed prolonged, through S period, large telomere replication. Furthermore, revealed ReD-FISH asymmetric signals were probably caused by partial replication of telomeres within an hour of 5-bromodeoxyuridine treatment due to the large size and special organization. We also found that in contrast to the telomeric halo from primary fibroblasts of bovine, mink, and common shrew, telomere halo of S. granarius consists of multiple loops bundled together, some of which contain rDNA. Here, we suggested several replicons firing possibly stochastic in each large telomere. Finally, we performed the TIF assay to reveal DNA damage responses at the telomeres, and along with TIF in nuclei, we found large bodies of telomeric DNA and ?-H2AX in the cytoplasm and on the surface of fibroblasts. We discuss the possibility of additional origin activation together with recombination-dependent replication pathways, mainly homologous recombination including BIR for replication fork stagnation overcoming and further S. granarius large telomere replication.     

Identification of the Determinant Conferring Permissive Substrate Usage in the Telomere Resolvase, ResT

Linear genome stability requires specialized telomere replication and protection mechanisms. A common solution to this problem in non-eukaryotes is the formation of hairpin telomeres by telomere resolvases (also known as protelomerases). These enzymes perform a two-step transesterification on replication intermediates to generate hairpin telomeres using an active site similar to that of tyrosine recombinases and type IB topoisomerases. Unlike phage telomere resolvases, the telomere resolvase from the Lyme disease pathogen Borrelia burgdorferi (ResT) is a permissive enzyme that resolves several types of telomere in vitro. However, the ResT region and residues mediating permissive substrate usage have not been identified. The relapsing fever Borrelia hermsii ResT exhibits a more restricted substrate usage pattern than B. burgdorferi ResT and cannot efficiently resolve a Type 2 telomere. In this study, we determined that all relapsing fever ResTs process Type 2 telomeres inefficiently. Using a library of chimeric and mutant B. hermsii/B. burgdorferi ResTs, we mapped the determinants in B. burgdorferi ResT conferring the ability to resolve multiple Type 2 telomeres. Type 2 telomere resolution was dependent on a single proline in the ResT catalytic region that was conserved in all Lyme disease but not relapsing fever ResTs and that is part of a 2-amino acid insertion absent from phage telomere resolvase sequences. The identification of a permissive substrate usage determinant explains the ability of B. burgdorferi ResT to process the 19 unique telomeres found in its segmented genome and will aid further studies on the structure and function of this essential enzyme.Replication and protection of telomeric DNA are required to ensure the genomic stability of all organisms with linear replicons. Until quite recently, it was assumed that linearity is a property confined to the replicons of eukaryotes and certain primarily eukaryotic viruses. However, a growing body of evidence indicates that linear DNA is also found in a broad range of bacteriophages (1–6) and in bacteria themselves (7–10), including the Borrelia species that cause Lyme disease and relapsing fever (11, 12). A common solution to the end replication and protection problem in non-eukaryotes is the covalent sealing of DNA ends in the form of hairpins (2, 4–6, 10, 11, 13–16). Hairpin DNA is not recognized as a double-strand break, and continuous synthesis of DNA around the hairpin loop abolishes the end replication problem. However, mother and daughter replicons are covalently linked at the junction of their telomeres following DNA replication; separation of the two replicons and formation of new hairpin telomeres require a DNA breakage and reunion process referred to as telomere resolution (17, 18).Resolution of the linear chromosome and plasmids in Borrelia species and of the linear plasmid prophages from Escherichia coli, Yersinia enterocolitica, and Klebsiella oxytoca is performed by telomere resolvases (also referred to as protelomerases) (5, 19–21). A growing number of candidate telomere resolvases have been identified in the genomes of eukaryotic viruses, phages, and bacteria (22, 23). Telomere resolvases are DNA cleavage and rejoining enzymes related to tyrosine recombinases and type 1B topoisomerases (19, 21, 22, 24, 25). Telomere resolvase catalyzes a two-step transesterification reaction in which staggered cuts are introduced 6 bp apart on either side of the axis of symmetry in the replicated telomere substrate (5, 19, 21, 24). Cleavage is accompanied by the formation of a 3′-phosphotyrosyl protein-DNA linkage. Subsequent nucleophilic attack on opposing strands by the free 5′-OH groups in the nicked substrate creates covalently closed hairpin telomeres. A recent crystal structure of the Klebsiella phage telomere resolvase (TelK) in complex with its substrate identified the residues involved in catalysis (25); all but one of these residues are conserved in all telomere resolvases (22), implying that the basic catalytic mechanism underlying telomere resolution is conserved. However, telomere resolvase sequences vary substantially outside of the central catalytic region (25, 26), and the enzymes characterized to date demonstrate important differences in substrate usage that likely reflect functionally distinct mechanisms of substrate interaction.The Borrelia burgdorferi telomere resolvase, ResT, appears to be particularly divergent. It is substantially smaller than phage telomere resolvases, and unlike its phage counterparts (5, 20, 21), it cannot efficiently resolve negatively supercoiled DNA (19, 27), presumably reflecting differences in the substrates resolved by phage and Borrelia telomere resolvases in vivo. On the other hand, B. burgdorferi ResT can fuse hairpin telomeres in a reversal of the resolution reaction (28), a function that is not shared with the phage telomere resolvase TelK (25). It can also synapse replicated telomeres and catalyze the formation of Holliday junctions (29). The ability of ResT to promote hairpin fusion has been proposed as the mechanism underlying the ongoing genetic rearrangements that are a prominent feature of the B. burgdorferi genome (18, 28). Finally, B. burgdorferi ResT can tolerate a surprising amount of variation in its substrate (30, 31), a feature that is not shared by phage telomere resolvases (21). Although B. burgdorferi ResT appears to be more permissive with a greater scope of activities than other telomere resolvases, the sequences mediating most of its unique properties have not yet been identified.The B. burgdorferi genome contains a total of 19 distinct hairpin sequences, all of which must be resolved by ResT (31). These sequences can be classified into three groups based on the presence and positioning of the box 1 motif, which is a critical determinant of activity in phage and Borrelia telomere resolvases (see Fig. 1A) (21, 24, 30). A box 1-like motif is also found in many of the hairpin telomeres sequenced to date (6, 14, 32–35), although its function in telomere resolution is unknown. The box 1 consensus sequence (TAT(a/t)AT) closely resembles the −10/Pribnow box and TATA box consensus sequences of prokaryotic and eukaryotic promoters (TATAAT and TATA(a/t)A(a/t), respectively), which undergo transient deformations that predispose them to melting (36) and are intrinsically bent and anisotropically flexible (37). Therefore, box 1 may facilitate nucleation of hairpin folding and/or may confer an intrinsic bend or flexibility to substrates that is important for the resolution reaction.Open in a separate windowFIGURE 1.Species-specific resolution of Type I and 2 telomeres. A, a schematic showing the three types of hairpin telomere found on the linear replicons of the B. burgdorferi genome (see Ref. 31). The box 1 sequence in Type 1 and 2 telomeres is situated 1 and 4 nucleotides away from the axis of symmetry, respectively, whereas Type 3 telomeres contain no clear box 1. B, a schematic illustrating the telomere resolution reaction substrate and products is shown along with two ethidium bromide-stained agarose gels showing telomere resolution assays. The gels show resolution kinetics for B. burgdorferi and B. hermsii ResT on Type 1 and 2 telomeres (plasmid substrates pYT1/lp17L and pYT92/chromL, respectively).B. burgdorferi ResT can resolve telomeres in which box 1 is located at positions 1 and 4 nucleotides away from the axis of symmetry (Type 1 and 2 telomeres, respectively), as well as AT-rich telomeres without a box 1 sequence (Type 3 telomeres) (see Fig. 1A) (30, 31). B. burgdorferi ResT cleaves telomeres at a fixed position relative to the axis of symmetry, independent of the location of box 1 (30). Positioning of the enzyme for cleavage in all telomere types is most likely driven by sequence-specific interactions between ResT domains 2 (catalytic) and/or 3 (C-terminal) and a fixed element upstream of box 1 that is positioned 14 nucleotides from the axis of symmetry in all Borrelia telomeres (box 3 and adjacent nucleotides) (see Figs. 1A and ​and2)2) (26, 30, 31). In contrast, box 1 and axis-flanking nucleotides are not involved in high affinity and/or sequence-specific interactions with ResT and require the ResT N-terminal domain for full protection in DNase footprinting assays (26, 27). The most likely candidate for interactions with box 1 and axis-flanking nucleotides is a Borrelia-specific hairpin-binding region in the N terminus, which is thought to promote a pre-hairpinning step involving strand opening at the axis (38).Open in a separate windowFIGURE 2.Alignment of 11 Borrelia ResT sequences. Shown is ClustalW2 alignment of ResT amino acid sequences from five Lyme disease Borrelia species (B. afzelii, B. spielmanii, B. valaisiana, B. garinii, and B. burgdorferi), five relapsing fever Borrelia species (B. turicatae, B. parkeri, B. hermsii, B. recurrentis, and B. duttonii), and one avian Borrelia species (B. anserina) (generated using ClustalW2 from the EBI web site) (19, 39–42, 48, 49). The sequences for B. anserina, B. parkeri, and B. turicatae ResTs are reported for the first time in this study (respective GenBank accession numbers are {"type":"entrez-nucleotide","attrs":{"text":"FJ882620","term_id":"242263541","term_text":"FJ882620"}}FJ882620, {"type":"entrez-nucleotide","attrs":{"text":"FJ882621","term_id":"242263543","term_text":"FJ882621"}}FJ882621, and {"type":"entrez-nucleotide","attrs":{"text":"FJ882623","term_id":"242263547","term_text":"FJ882623"}}FJ882623). Sequences are arranged in order of similarity to neighboring sequences and are colored in JalView using the Zappo coloring scheme for identifying amino acids with similar physicochemical properties (50). Only residues that are identical in 100% of ResTs are indicated by colored shading. Arrows above the alignment indicate ResT domain boundaries identified by chymotrypsin digest, sequence comparison with other proteins, and HHsenser predictions (26, 51). The hairpin-binding motif found in cut-and-paste transposases is indicated beneath the alignment by white text on a black background (38). The positions corresponding to the active site residues in tyrosine recombinases, type IB topoisomerases, and TelK are indicated by blue asterisks below the sequence, with the active site tyrosine nucleophile at position 335 marked by a red asterisk (22, 25). The ringed black dot below position 326 indicates an amino acid in the active site region that differs in Lyme disease and relapsing fever ResTs. Sequences above the black line drawn between B. burgdorferi and B. turicatae are from Lyme disease Borrelia species; sequences below the black line are from relapsing fever Borrelia species. The ResT sequence from the avian Borrelia species B. anserina is shown at bottom.ResT from the relapsing fever Borrelia species Borrelia hermsii exhibits a more restricted substrate usage pattern in vitro when compared with ResT from the Lyme disease pathogen B. burgdorferi (39). Specifically, B. hermsii ResT is unable to efficiently resolve a Type 2 telomere. Therefore, B. burgdorferi ResT appears to be a more permissive enzyme than its relapsing fever counterpart. In this study, we investigated the basis for permissive substrate usage by B. burgdorferi ResT. Using a library of chimeric B. hermsii/B. burgdorferi ResTs, we mapped the sequence determinants in B. burgdorferi ResT that confer the ability to resolve multiple Type 2 telomeres. Surprisingly, this approach indicated that Type 2 telomere resolution was crucially regulated by a single proline residue located in a small Borrelia-specific insertion in the central catalytic region of ResT. The proline at this position was conserved in the ResTs from all Lyme disease Borrelia species but in none of the ResTs from relapsing fever Borrelia species, which were unable to efficiently resolve Type 2 telomeres in vitro. This study has identified a specific residue in ResT responsible for permissive substrate usage patterns.