The telomere resolvase, TelA, utilizes an underwound pre-cleavage intermediate to promote hairpin telomere formation
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ABSTRACT:
SUBMITTER: Balouchi M
PROVIDER: S-EPMC10686437 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description: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.
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Project description: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.
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Project description: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.
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Project description:The first steps of poxvirus DNA synthesis yield concatemeric arrays of covalently linked genomes. The virus-encoded Holliday junction resolvase is required to process concatemers into unit-length genomes for packaging. Previous studies of the vaccinia virus resolvase have been problematic due to poor protein solubility. We found that fowlpox virus resolvase was much more tractable. Fowlpox resolvase formed complexes with a variety of branched DNA substrates, but not linear DNA, and had the highest affinity for a Holliday junction substrate, illustrating a previously unappreciated affinity for Holliday junctions over other substrates. The cleavage activity was monitored in fixed time assays, showing that, as with vaccinia resolvase, the fowlpox enzyme could cleave a wide array of branched DNA substrates. Single turnover kinetic analysis revealed the Holliday junction substrate was cleaved 90-fold faster than a splayed duplex substrate containing a single to double strand transition. Multiple turnover kinetic analysis, however, showed that the cleavage step was not limiting for the full reaction cycle. Cleavage by resolvase was also tightly coupled at symmetrical positions across the junction, and coupling required the complete Holliday junction structure. Last, we found that cleavage of an extruded cruciform yielded a product, which after treatment with ligase, had the properties expected for covalently closed DNA hairpin ends, as is seen for poxvirus genome monomers. These findings provide a tractable poxvirus resolvase usable for the development of small molecule inhibitors.
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Project description: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.
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Project description: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.
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