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.

The Borrelia burgdorferi telomere resolvase, ResT, possesses ATP-dependent DNA unwinding activity.

Spirochetes of the genus Borrelia possess unusual genomes harboring multiple linear and circular replicons. The linear replicons are terminated by covalently closed hairpin (hp) telomeres. Hairpin telomeres are formed from replicated intermediates by the telomere resolvase, ResT, in a phosphoryl transfer reaction with mechanistic similarities to those promoted by type 1B topoisomerases and tyrosine recombinases. There is growing evidence that ResT is multifunctional. Upon ResT depletion DNA replication unexpectedly ceases. Additionally, ResT possesses RecO-like biochemical activities being able to promote single-strand annealing on both free ssDNA and ssDNA complexed with cognate single-stranded DNA binding protein. We report here that ResT possesses DNA-dependent ATPase activity that promotes DNA unwinding with a 3΄-5΄ polarity. ResT can unwind a variety of substrates including synthetic replication forks and D-loops. We demonstrate that ResT's twin activities of DNA unwinding and annealing can drive regression of a model replication fork. These properties are similar to those of the RecQ helicase of the RecF pathway involved in DNA gap repair. We propose that ResT's combination of activities implicates it in replication and recombination processes operating on the linear chromosome and plasmids of Borrelia burgdorferi.

The Borrelia burgdorferi telomere resolvase, ResT, anneals ssDNA complexed with its cognate ssDNA-binding protein.

Spirochetes of the genus Borrelia possess unusual genomes that consist in a linear chromosome and multiple linear and circular plasmids. The linear replicons are terminated by covalently closed hairpin ends, referred to as hairpin telomeres. The hairpin telomeres represent a simple solution to the end-replication problem. Deoxyribonucleic acid replication initiates internally and proceeds bidirectionally toward the hairpin telomeres. The telomere resolvase, ResT, forms the hairpin telomeres from replicated telomere intermediates in a reaction with similarities to those promoted by type IB topoisomerases and tyrosine recombinases. ResT has also been shown to possess DNA single-strand annealing activity. We report here that ResT promotes single-strand annealing of both free DNA strands and ssDNA complexed with single-stranded DNA binding protein (SSB). The annealing of complementary strands bound by SSB requires a ResT-SSB interaction that is mediated by the conserved amphipathic C-terminal tail of SSB. These properties of ResT are similar to those demonstrated for the recombination mediator protein, RecO, of the RecF pathway. Borrelia burgdorferi is unusual in lacking identifiable homologs of the RecFOR proteins. We propose that ResT may provide missing RecFOR functions.

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.

Structure, function, and evolution of linear replicons in Borrelia.

Spirochetes of the genus Borrelia include important human pathogens that cause Lyme borreliosis and relapsing fever. The genomes of Borrelia species can be composed of up to 24 DNA molecules, most of which are linear. The plasmid content and linear replicon sequence arrangement vary widely between isolates. The linear replicons are terminated by covalently closed DNA hairpins or hairpin telomeres. Replication of these elements involves a unique reaction, called telomere resolution, to produce hairpin telomeres from replicative intermediates. The telomere resolvase, ResT, is thought to contribute to the genetic flux of the linear molecules by promoting stabilized telomere fusions. Telomere resolvases are related to the tyrosine recombinases and ResT can generate the crucial reaction intermediate of this class of enzyme, the Holliday junction. This observation has led to the proposal that telomere resolvases evolved from tyrosine recombinases inducing DNA linearization in the genomes that acquired them.

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.

Design and characterization of hyperactive mutants of the Agrobacterium tumefaciens telomere resolvase, TelA.

Telomere resolvases are a family of DNA cleavage and rejoining enzymes that produce linear DNAs terminated by hairpin telomeres from replicated intermediates in bacteria that possess linear replicons. The telomere resolvase of Agrobacterium tumefaciens, TelA, has been examined at the structural and biochemical level. The N-terminal domain of TelA, while not required for telomere resolution, has been demonstrated to play an autoinhibitory role in telomere resolution, conferring divalent metal responsiveness on the reaction. The N-terminal domain also inhibits the competing reactions of hp telomere fusion and recombination between replicated telomere junctions. Due to the absence of the N-terminal domain from TelA/DNA co-crystal structures we produced an AlphaFold model of a TelA monomer. The AlphaFold model suggested the presence of two inhibitory interfaces; one between the N-terminal domain and the catalytic domain and a second interface between the C-terminal helix and the N-core domain of the protein. We produced mutant TelA's designed to weaken these putative interfaces to test the validity of the modeled interfaces. While our analysis did not bear out the details of the predicted interfaces the model was, nonetheless, extremely useful in guiding design of mutations that, when combined, demonstrated an additive activation of TelA exceeding 250-fold. For some of these hyperactive mutants stimulation of telomere resolution has also been accompanied by activation of competing reactions. However, we have also characterized hyperactive TelA mutants that retain enough autoinhibition to suppress the competing reactions.

The telomere resolvase, TelA, utilizes an underwound pre-cleavage intermediate to promote hairpin telomere formation.

The telomere resolvase, TelA, forms the hairpin telomeres of the linear chromosome of Agrobacterium tumefaciens in a process referred to as telomere resolution. Telomere resolution is a unique DNA cleavage and rejoining reaction that resolves replicated telomere junctions into a pair of hairpin telomeres. Telomere resolvases utilize a reaction mechanism with similarities to that of topoisomerase-IB enzymes and tyrosine recombinases. The reaction proceeds without the need for high-energy cofactors due to the use of a covalent, enzyme-cleaved DNA intermediate that stores the bond energy of the cleaved bonds in 3'-phosphotyrosyl linkages. The cleaved DNA strands are then refolded into a hairpin conformation and the 5'-OH ends of the refolded strands attack the 3'-phosphotyrosine linkages in order to rejoin the DNA strands into hairpin telomeres. Because this kind of reaction mechanism is, in principle, reversible it is unclear how TelA controls the direction of the reaction and propels the reaction to completion. We present evidence that TelA forms and/or stabilizes a pre-cleavage intermediate that features breakage of the four central basepairs between the scissile phosphates prior to DNA cleavage to help propel the reaction forwards, thus preventing abortive cleavage and rejoining cycles that regenerate the substrate DNA. We identify eight TelA sidechains, located in the hairpin-binding module and catalytic domains of TelA, implicated in this process. These mutants were deficient for telomere resolution on parental replicated telomere junctions but were rescued by introduction of substrate modifications that mimic unwinding of the DNA between the scissile phosphates.

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.

Single stranded DNA annealing is a conserved activity of telomere resolvases.

Bacterial species of the genera Agrobacterium and Borrelia possess chromosomes terminated by hairpin telomeres. Replication produces dimeric replication intermediates fused via replicated telomere junctions. A specialized class of enzymes, referred to as telomere resolvases, promotes the resolution of the replicated intermediate into linear monomers terminated by hairpin telomeres. Telomere resolution is catalyzed via DNA cleavage and rejoining events mechanistically similar to those promoted by topoisomerase-IB and tyrosine recombinase enzymes. Examination of the borrelial telomere resolvase, ResT, revealed unanticipated multifunctionality; aside from its expected telomere resolution activity ResT possessed a singled-stranded DNA (ssDNA) annealing activity that extended to both naked ssDNA and ssDNA complexed with its cognate single-stranded DNA binding protein (SSB). At present, the role this DNA annealing activity plays in vivo remains unknown. We have demonstrated here that single-stranded DNA annealing is also a conserved property of the agrobacterial telomere resolvase, TelA. This activity in TelA similarly extends to both naked ssDNA and ssDNA bound by its cognate SSB. TelA's annealing activity was shown to stem from the N-terminal domain; removal of this domain abolished annealing without affecting telomere resolution. Further, independent expression of the N-terminal domain of TelA produced a functional annealing protein. We suggest that the apparent conservation of annealing activity in two telomere resolvases, from distantly related bacterial species, implies a role for this activity in hairpin telomere metabolism. Our demonstration of the separation of the telomere resolution and annealing activities of TelA provides a platform for future experiments aimed at identifying the role DNA annealing performs in vivo.

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 (rTels) 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.

Biochemical characterization of Borrelia burgdorferi's RecA protein.

RecA plays key roles in DNA recombination, replication and repair. Mutation of recA in the Lyme disease spirochete, Borrelia burgdorferi, fails to produce some of the phenotypes expected from study of recA mutation in other organisms. 'Missing' recA phenotypes include a lack of growth or viability effects, including in the presence of DNA damage, and a lack of a role in vlsE antigenic variation and infectivity. We present a purification and biochemical characterization of recombinant B. burgdorferi RecA protein. We find that B. burgdorferi RecA displays the expected properties of being a DNA-dependent ATPase, of having an intrinsic binding preference for ssDNA over dsDNA enhanced by ATP binding, of promoting DNA pairing and strand exchange reactions and of having a detectable coprotease activity with E. coli LexA repressor. DNA pairing and strand exchange reactions promoted by B. burgdorferi RecA show an unusually strong dependence upon the presence of the cognate ssDNA binding protein (SSB) but are very sensitive to inhibition by SSB when the ssDNA was prebound by SSB. This indicates B. burgdorferi RecA may have an enhanced requirement for recombinational mediators to promote RecA-SSB exchange, despite the absence of homologues of the RecF pathway proteins that normally play this role in eubacteria. Finally, we do not find any unusual, intrinsic properties of B. burgdorferi's RecA protein to explain the unusual phenotype of recA mutation and suggest that there may be alternative recombinase functions that could explain the 'missing' phenotypes.

Hairpin Telomere Resolvases.

Covalently closed hairpin ends, also known as hairpin telomeres, provide an unusual solution to the end replication problem. The hairpin telomeres are generated from replication intermediates by a process known as telomere resolution. This is a DNA breakage and reunion reaction promoted by hairpin telomere resolvases (also referred to as protelomerases) found in a limited number of phage and bacteria. The reaction promoted by these enzymes is a chemically isoenergetic two-step transesterification without a requirement for divalent metal ions or high-energy cofactors and uses an active site and mechanism similar to that for type IB topoisomerases and tyrosine recombinases. The small number of unrelated telomere resolvases characterized to date all contain a central, catalytic core domain with the active site, but in addition carry variable C- and N-terminal domains with different functions. Similarities and differences in the structure and function of the telomere resolvases are discussed. Of particular interest are the properties of the Borrelia telomere resolvases, which have been studied most extensively at the biochemical level and appear to play a role in shaping the unusual segmented genomes in these organisms and, perhaps, to play a role in recombinational events.

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.

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.

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.

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.

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.

The Mu three-site synapse: a strained assembly platform in which delivery of the L1 transposase binding site triggers catalytic commitment.

The Mu DNA transposition reaction proceeds through a three-site synaptic complex (LER), including the two Mu ends and the transpositional enhancer. We show that the LER contains highly stressed DNA regions in the enhancer and in the L1 transposase binding site. We propose that the L1 site acts as the keystone for assembly of a catalytically competent transpososome. Delivery of L1 through HU-mediated bending completes LER assembly, provides the trigger for necessary conformational transitions in transpososome formation, and allows target capture to occur. Relief of the stress at L1 and the enhancer may help drive Mu A tetramerization and engagement of the Mu ends by the transposase active site.