The telomere resolvase of the Lyme disease spirochete,Borrelia burgdorferi,promotes DNA single-strand annealing and strand exchange

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 hairpin telomeres. Hairpin telomeres present an uninterrupted DNA chain to the replication machinery overcoming the ‘end-replication problem’ for the linear replicons. Hairpin telomeres are formed from inverted repeat replicated telomere junctions by the telomere resolvase, ResT. ResT uses a reaction mechanism similar to that of the type IB topoisomerases and tyrosine recombinases. We report here that ResT also possesses single-strand annealing activity and a limited ability to promote DNA strand exchange reactions on partial duplex substrates. This combination of activities suggests ResT is a nexus between the seemingly distinct processes of telomere resolution and homologous recombination. Implications for hairpin telomere replication and linear plasmid recombination, including antigenic variation, are discussed.     

The N-terminal domain of the Agrobacterium tumefaciens telomere resolvase,TelA, regulates its DNA cleavage and rejoining activities

Linear replicons can be found in a minority of prokaryotic organisms, including Borrelia species and Agrobacterium tumefaciens. The problem with replicating the lagging strand end of linear DNAs is circumvented in these organisms by the presence of covalently closed DNA hairpin telomeres at the DNA termini. Telomere resolvases are enzymes responsible for generating these hairpin telomeres from a dimeric replication intermediate through a two-step DNA cleavage and rejoining reaction referred to as telomere resolution. It was previously shown that the agrobacterial telomere resolvase, TelA, possesses ssDNA annealing activity in addition to telomere resolution activity. The annealing activity derives, chiefly, from the N-terminal domain. This domain is dispensable for telomere resolution. In this study, we used activity analyses of an N-terminal domain deletion mutant, domain add back experiments, and protein–protein interaction studies and we report that the N-terminal domain of TelA is involved in inhibitory interactions with the remainder of TelA that are relieved by the binding of divalent metal ions. We also found that the regulation of telomere resolution by the N-terminal domain of TelA extends to suppression of inappropriate enzymatic activity, including hairpin telomere fusion (reaction reversal) and recombination between replicated telomeres to form a Holliday junction.     

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.     

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.     

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.     

Catalytic residues of the telomere resolvase ResT: a pattern similar to, but distinct from, tyrosine recombinases and type IB topoisomerases

ResT is a member of the telomere resolvases, a newly discovered class of DNA breakage and reunion enzymes. These enzymes are involved in the formation of co-valently closed hairpin DNA ends that are found in linear prokaryotic chromosomes and plasmids. The hairpins are generated by telomere resolution, where the replicated linear DNA ends are processed by DNA breakage followed by joining of DNA free ends to the complementary strand of the same molecule. Previous studies have shown that ResT catalyzes hairpin formation through a two-step transesterification similar to tyrosine recombinases and type IB topoisomerases. In the present study we have probed the reaction mechanism of ResT. The enzyme was found to efficiently utilize a substrate with a 5'-bridging phosphorothiolate at each cleavage site, similar to tyrosine recombinases/type IB topoisomerases. Using such a substrate to trap the covalent protein-DNA intermediate, coupled with affinity purification and mass spectroscopy, we report a new, non-radioactive approach to directly determine the position of the amino acid in the protein, which is linked to the DNA. We report that tyrosine 335 is the active site nucleophile in ResT, strengthening the link between ResT and tyrosine recombinases/type IB topoisomerases. However, a distinct pattern of catalytic residues with similarities, but distinct differences from the above enzymes was suggested. The differences include the apparent absence of a general acid catalyst, as well as the dispensability of the final histidine in the RKHRHY hexad. Finally, two signature motifs (GRR(2X)E(6X)F and LGH(4-6X)T(3X)Y) near the catalytic residues of aligned telomere resolvases are noted.     

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.     

Unexpected twist: harnessing the energy in positive supercoils to control telomere resolution

Negative DNA supercoiling is an important conformational property of bacterial DNA that plays a significant role in a wide variety of DNA transactions. In contrast, positive DNA supercoiling is a by-product of cellular processes that involve helical unwinding or movement of DNA by a fixed translocase, and has generally been considered a necessary evil requiring removal. We now report the first evidence suggesting a physiological role for positive supercoiling; this occurs in telomere resolution in the related Lyme disease and relapsing fever Borrelia spirochetes. Telomere resolution is the process whereby covalently closed hairpin telomeres are generated from replicative intermediates by the telomere resolvase, ResT. We observe a 20-fold and greater stimulation of the reaction by positive supercoiling, which facilitates formation of a previously unobserved reaction intermediate. Our data suggest the possibility that the free energy of positive supercoiling, a resource with no previously described cellular function, may be harnessed and utilized as a regulator of post-replication events.     

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.     

Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation

Escherichia coli phage P1 Cre recombinase catalyzes the site-specific recombination of DNA containing loxP sites. We report here two crystal structures of a wild-type Cre recombinase–loxP synaptic complex corresponding to two distinct reaction states: an initial pre-cleavage complex, trapped using a phosphorothioate modification at the cleavable scissile bond that prevents the recombination reaction, and a 3′-phosphotyrosine protein–DNA intermediate resulting from the first strand cleavage. In contrast to previously determined Cre complexes, both structures contain a full tetrameric complex in the asymmetric unit, unequivocally showing that the anti-parallel arrangement of the loxP sites is an intrinsic property of the Cre–loxP recombination synapse. The conformation of the spacer is different to the one observed for the symmetrized loxS site: a kink next to the scissile phosphate in the top strand of the pre-cleavage complex leads to unstacking of the TpG step and a widening of the minor groove. This side of the spacer is interacting with a ‘cleavage-competent’ Cre subunit, suggesting that the first cleavage occurs at the ApT step in the top strand. This is further confirmed by the structure of the 3′-phosphotyrosine intermediate, where the DNA is cleaved in the top strands and covalently linked to the ‘cleavage-competent’ subunits. The cleavage is followed by a movement of the C-terminal part containing the attacking Y324 and the helix N interacting with the ‘non-cleaving’ subunit. This rearrangement could be responsible for the interconversion of Cre subunits. Our results also suggest that the Cre-induced kink next to the scissile phosphodiester activates the DNA for cleavage at this position and facilitates strand transfer.     

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.     

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.     

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.     

Resolution of parvovirus dimer junctions proceeds through a novel heterocruciform intermediate

The minute virus of mice initiator protein, NS1, excises new copies of the left viral telomere in a single sequence orientation, dubbed flip, during resolution of the junction between monomer genomes in palindromic dimer intermediate duplexes. We examined this reaction in vitro using both (32)P-end-labeled linear substrates and similar unlabeled templates labeled by incorporation of [alpha-(32)P]TTP during the synthesis. The observed products suggest a resolution model that explains conservation of the hairpin sequence and in which a novel heterocruciform intermediate plays a crucial role. In vitro, NS1 initiates two replication pathways from OriL(TC), the single active origin embedded in one arm of the dimer junction. NS1-mediated nicking liberates a base-paired 3' nucleotide to prime DNA synthesis and, in a reaction we call "read-through synthesis," forks established while the substrate is a linear duplex synthesize DNA in the flop orientation, leading to DNA amplification but not to junction resolution. Nicking leaves NS1 covalently attached to the 5' end of the DNA, where it can serve as a 3'-to-5' helicase, unwinding the NS1-associated strand. In the second pathway, resolution substrates are created when such unwinding induces the palindrome to reconfigure into a cruciform prior to fork assembly. New forks can then synthesize DNA in the flip orientation, copying one cruciform arm and creating a heterocruciform intermediate. Resolution proceeds via hairpin transfer in the extended arm of the heterocruciform, which releases one covalently closed duplex telomere and a partially single-stranded junction intermediate. We suggest that the latter intermediate is finally resolved via an NS1-induced single-strand nick at the otherwise inactive origin, OriL(GAA).     

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.     

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site‐specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′‐phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg‐308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti‐hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site‐specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns.     

Unwinding forward and sliding back: an intermittent unwinding mode of the BLM helicase

There are lines of evidence that the Bloom syndrome helicase, BLM, catalyzes regression of stalled replication forks and disrupts displacement loops (D-loops) formed during homologous recombination (HR). Here we constructed a forked DNA with a 3′ single-stranded gap and a 5′ double-stranded handle to partly mimic a stalled DNA fork and used magnetic tweezers to study BLM-catalyzed unwinding of the forked DNA. We have directly observed that the BLM helicase may slide on the opposite strand for some distance after duplex unwinding at different forces. For DNA construct with a long hairpin, progressive unwinding of the hairpin is frequently interrupted by strand switching and backward sliding of the enzyme. Quantitative study of the uninterrupted unwinding length (time) has revealed a two-state-transition mechanism for strand-switching during the unwinding process. Mutational studies revealed that the RQC domain plays an important role in stabilizing the helicase/DNA interaction during both DNA unwinding and backward sliding of BLM. Especially, Lys1125 in the RQC domain, a highly conserved amino acid among RecQ helicases, may be involved in the backward sliding activity. We have also directly observed the in vitro pathway that BLM disrupts the mimic stalled replication fork. These results may shed new light on the mechanisms for BLM in DNA repair and homologous recombination.     

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.