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

Shu Hui Huang; Kayla Abrametz; Siobhan L McGrath; Kerri Kobryn

doi:10.1371/journal.pone.0307590

. 2024 Jul 25;19(7):e0307590. doi: 10.1371/journal.pone.0307590

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

Shu Hui Huang ¹, Kayla Abrametz ¹, Siobhan L McGrath ^1,^¤, Kerri Kobryn ^1,^*

Editor: Arthur J Lustig²

PMCID: PMC11271964 PMID: 39052566

Abstract

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.

Introduction

Spirochetes of the Borrelia genus and several species of Agrobacterium possess linear replicons terminated by covalently closed hairpin (hp) telomeres. The replication cycle of these linear DNAs is thought to involve initiation of replication at an internal origin that sends out a replication fork towards each hp telomere [1–3]. As replication is completed the hp telomeres are converted into replicated telomere junctions (rTel) present in an inverted repeat circular dimer. This dimer is resolved into a pair of linear DNAs terminated by the hp telomeres by a DNA cleavage and rejoining reaction referred to as telomere resolution ([4–6]; S1 Fig in S1 File). Telomere resolvases are a unique family of DNA cleavage and rejoining enzymes that promote telomere resolution at the rTel junctions. Four exemplars of the telomere resolvase family have been well characterized either by biochemical, genetic or structural means: TelA from Agrobacterium tumefaciens, ResT from Borrelia burgdorferi, TelK from the Klebsiella phage ϕKO2 and TelN from the E. coli phage N15 [7–10]. The telomere resolvases promote a reaction with mechanistic similarities to those promoted by type IB topoisomerases and tyrosine recombinases, utilizing a catalytic domain with structural similarity to the catalytic domains of these enzymes [10].

TelA, from Agrobacterium tumefaciens, has been characterized both biochemically and structurally [9, 11, 12]. Aside from promoting telomere resolution, TelA has also been reported to promote single-stranded DNA annealing reactions with both naked ssDNA and ssDNA complexed with its cognate single-stranded DNA binding protein [13]. These findings mirror those found for the B. burgdorferi telomere resolvase, ResT [14, 15]. The N-terminal domain of TelA is required for this activity but appears to be dispensable for telomere resolution [11, 16]. A TelA mutant with an N-terminal domain truncation can still promote telomere resolution but has lost much of the responsiveness to the presence of divalent metal ions (Mg²⁺ or Ca²⁺) normally observed with wild type TelA. The stimulatory effect of divalent metal ions appears to relieve autoinhibition by the N-terminal domain by affecting a step after substrate DNA binding [16]. This latter study suggested that the N-terminal domain also acts to restrain competing reactions that telomere resolvases have been shown to be capable of promoting: the reverse reaction of hp telomere fusion and the side reaction of generating Holliday junctions (HJ) via strand exchange between rTels [16–18]. A similar autoinhibitory role for the ResT N-terminal domain has been demonstrated that operates to mask substrate binding; rendering telomere resolution responsive to positive DNA supercoiling [19, 20].

In this study we used an AlphaFold2 generated model of a TelA monomer unbound to DNA to search for potential autoinhibitory interfaces in TelA responsible for regulating the activity of TelA. This modeling exercise identified two potential interfaces: one between the N-terminal domain and the catalytic domain encompassing residues 5–9 and residues 333–337; a second interface was potentially identified between the C-terminal helix, residues 421–442 and the hairpin-binding module helix 198–216 with charged residues R440/K441 potentially interacting with the D202 residue at the top of the hairpin binding module of TelA. By the use of targeted deletions and point mutations designed to weaken or abolish these interfaces we tested the model. We find that the model was mostly incorrect in its detailed predictions but was, nonetheless, extremely useful in guiding selection of mutations, that when combined, produced a telomere resolvase that was more than 250-fold activated for telomere resolution and that was largely independent of divalent metal ions.

Materials and methods

DNAs

All synthetic DNAs used to assemble substrates or produce site-directed mutations were purchased from Integrated DNA Technologies (IDT) and are listed in S1 Table in S1 File. All telomere resolution assays to derive initial rates used SspI-linearized pEKK392, the construction of which is reported elsewhere [16]. Telomere resolution reactions with a mutant rTel that has a CCATGA sequence between the scissile phosphates that inhibits hp formation, instead of a TCATGA wild type sequence, were conducted with pEKK495. pEKK495’s assembly is reported elsewhere [12].

Proteins

All TelA mutants reported in this study were generated by site-directed mutagenesis using oligonucleotides listed in S1 Table in S1 File. The induction and expression conditions are listed in S2 Table in S1 File. All TelA purifications proceeded as reported elsewhere, except with induction and expression conditions listed in S2 Table in S1 File [13, 16].

Telomere resolution assays

Telomere resolution assays to derive initial rates of reaction were incubated at 0°C, unless otherwise stated in the Figures and their legends. This reaction temperature ensured that we were able to measure rates for highly activated mutants. The reaction buffer contained 25 mM HEPES (pH 7.6), 1 mM DTT, 100 μg/mL BSA, 50 mM potassium glutamate, 1.8 μg/mL SspI linearized pEKK392 substrate DNA, 76 nM TelA and either 1 mM EDTA or 2 mM CaCl₂. A 120 μL master reaction was used. 18 μL aliquots were withdrawn at the indicated timepoints and the reaction was terminated by resuspension of the aliquots into SDS-containing load dye to a 1X concentration. 1X load dye contains 0.2% SDS, 20 mM EDTA, 3.2% glycerol, and 0.024% bromophenol blue. Subsequently, the timepoint samples were loaded to 0.8% agarose 1X TAE gels that were electophoresed at 3V/cm for 3 hours. The results were visualized by staining the gels with 0.5 μg/mL ethidium bromide for 30 min, followed by destaining in distilled water for 30 min. TelA (107–442), denoted as ΔN in the Figure legends, was found to be inactive at 0°C, so the initial rate for this protein was determined in reactions performed as noted above, excepting that a 30°C incubation temperature was utilized. Where addition of the N-terminal domain was added in trans, TelA (1–106), denoted as N, was added at a concentration of 800 nM. In order to assess the ability of TelA mutants to process a mutant rTel with a sequence (CCATGA as opposed to TCATGA found in wild type) between the scissile phosphates that inhibits hairpin formation we utilized reactions incubated at 30°C utilizing pEKK495. The ability of hyperactive TelA mutants to resolve negatively supercoiled substrate plasmid, normally a very poorly reactive substrate, was assessed by reactions incubated at 30°C for 60 min using 50 nM TelA and 2 μg/ml pEKK392. Gel images were visualized using a BioRad GelDoc system and the results were quantified with BioRad’s QuantityOne software. Graphs were generated with Prism’s GraphPad 6.0 software.

Half-site cleavage and hairpin fusion assays

Half-site cleavage and hairpin formation assays were performed in a buffer containing 25 mM HEPES (pH 7.6), 1 mM DTT, 2 mM CaCl₂, 100 μg/mL BSA and 50 mM potassium glutamate. 76 nM TelA was incubated with 5 nM of 5’ fluorescein-endlabeled parental half-site (OKBA27F/OGCB871) or the hp telomere (OKBA28F) at 30°C for the times indicated (F indicates the labeled strand). Mock versions of the half-site and hp that maintain the sequence composition of the bona fide substrates were tested as well using oligonucleotides (OKBA29F/OGCB913; mock rTel and OKBA30F; mock hp). A 60 μL reaction volume was used and 18 μL aliquots were removed at the indicated timepoints and resuspended into a 1X concentration of an SDS-containing load dye. The samples were loaded to 8% PAGE, 1X Tris-acetate, EDTA (TAE), 0.1% SDS gels and were electrophoresed on 20 x 20 cm gels at 20V/cm for 150 min. These gels are native for DNA but denaturing for protein. The SDS allows the TelA covalently linked to the cleaved DNA to enter the gel. Gel images were visualized using a BioRad GelDoc system and the results were quantified with BioRad’s QuantityOne software.

Site-specific recombination assays

The ability of TelA mutants to promote recombination between rTels was assayed using negatively supercoiled pEKK392 and a 5’-fluorescein end-labeled rTel. 190 nM of the indicated mutants were reacted with 10 μg/mL of pEKK392 and 70 nM oligonucleotide rTel (OGCB951F/OGCB952) in our standard telomere resolution reaction buffer with 2 mM CaCl₂ (see above). The oligonucleotide rTel was partitioned between 20 nM of fluorescein labeled oligonucleotide rTel and 50 nM of unlabeled rTel. Control incubations omitted TelA. Reactions were incubated at 30°C for 2 h followed by the addition of 5X SDS loading dye to a 1X final concentration prior to gel loading. Reactions were loaded to 0.8% (w/v) agarose 1X TAE gels and electrophoresed at 1V/cm for 19 h. The bottom of the gel with the free unreacted rTels were cut off and then gels were imaged for fluorescein labeled material. Subsequently, gels were stained with 0.5 μg/mL ethidium bromide and visualized on a Biorad GelDoc system using the ethidium bromide program to visualize the plasmid substrate and molecular weight markers.

Results

Modeling of potential autoinhibitory interfaces within TelA

There has been a co-crystal structure of TelA with various substrates and reaction products available for several years [9]. While extremely informative the structures, unfortunately, do not resolve the N-terminal domain or show a structure of the enzyme unbound to DNA. Since the remarkable improvement in structure modeling reliability represented by AlphaFold2 we were interested in modeling TelA as an unbound monomer to see if we could discover the molecular basis for the N-terminal domain’s autoinhibitory role on telomere resolution [16, 21]. The top ranked model generated by AlphaFold2 is shown in Fig 1A alongside a monomer view of TelA bound to a hairpin (hp) telomere. AlphaFold2 predicts the structure of the N-terminal domain (shown in red) and shows it to stack upon the catalytic domain of TelA (pink) in a manner that, potentially, could interfere with activity. Additionally, the C-terminal helix (shown in green) is predicted to switch from a conformation found in TelA bound to substrate or products in which the C-terminal helix is swapped between protomers of the active dimer to a conformation that interacts with the hairpin binding module helix of the TelA N-core domain in the monomer. This helix in the N-core domain has many critical residues needed for telomere resolution and could, therefore, represent another autoinhibitory interface [12, 22, 23]. Examination of the details of the interfaces in the AlphaFold2 model reveals the juxtaposition of oppositely charged residues across the interfaces (Fig 1B). This suggested that there may be two autoinhibitory interfaces in the TelA monomer maintained, in part, by electrostatic interactions across the interfaces. We focused on the potential electrostatic interactions, as this would allow us to employ the method of neutralizing and reversing charges and to map potential interacting pairs via charge reversals in double mutant contexts. The predicted aligned error plot for the model is presented in S2 Fig in S1 File and indicates a low confidence (~30 Å) in the prediction for interface 1 but much higher confidence for interface 2 (~10 Å).

Fig 1 — A) TelA-hairpin (hp) product complex structure (one protomer of the dimer shown) vs. an AlphaFold2 model of a TelA monomer without bound DNA. In the model the positively charged beginning of the N-terminal domain interacts with an acidic patch in the catalytic domain (interface 1) and the C-terminal helix reorients to form the autoinhibitory interface 2. The N-terminal domain (red) is encompassed by residues 1–106, the N-core domain (blue) by residues 107–217, the catalytic domain (pink) by residues 218–410 and the C-terminal helix (green) by residues 411–442. Within the N-core domain the hairpin binding module lies between residues 197–216 and comprised the long alpha helix that connects the N-core to the catalytic domain. The model derived from the structural data was generated using PyMol and PDB accession # 4e0g. The model derived from AlphaFold2 was generated using using PyMol and AlphaFold2 [21]. B) Details of the autoinhibitory interfaces with charged residues of interest shown.

Mutation of interfaces 1 & 2 stimulates telomere resolution

In order to investigate whether the model was predicting real autoinhibitory interfaces, we generated, expressed and purified TelA mutants designed to weaken or interrupt the hypothesized interfaces. To assess the effects of these mutations on TelA activity we measured the initial rate of reaction of each mutant under conditions without divalent metal ion (1mM EDTA) or in the presence of 2 mM Ca²⁺. We measured the rates at 0°C instead of the standard 30°C reaction temperature in order to slow reactions down sufficiently so that we could determine initial rates for hyperactive mutants.

We employed charge neutralization (to alanine), charge reversal or deletion of the charged residues at each interface (Fig 2). At interface 1 the N-terminal domain residues K5, R6 and K7 were examined by charge reversal mutations to D or E residues. This afforded modest stimulation (1.4–2.7-fold) of the reaction under conditions with Ca²⁺ present but were not stimulatory under conditions with EDTA (Fig 2A). We took these results to indicate that the mutations had weakened the interface enough to be apparent under metal-stimulation but not sufficiently to activate TelA in the absence of divalent metal ions. Amongst residues of interest in the N-terminal domain, mutation of the K9 residue to A or E led to the greatest stimulation of the reaction compared to wild type TelA, affording 7.7-fold and 3.9-fold stimulation, respectively, in conditions containing Ca²⁺. On the other side of interface 1 the D332, D333 and E337 residues were mutated to neutralize and/or reverse the charge of the residue. The D332A mutation was less active than wild type at 0°C and was, therefore, inferred not to contribute to the interface. D333A provided stimulation similar to that afforded by the K9A mutation and D333K still provided modest stimulation in conditions with Ca²⁺ (7.7 and 2.6-fold). The greatest stimulatory effect of single point mutations at interface 1 was found with mutation of E337 to A or K, affording 11.6-fold and 18.5-fold stimulation of the reaction rate with Ca²⁺, respectively. Interestingly, the calcium dependence of the reaction seems attenuated with the E337K mutant with a robust 18-fold stimulation of the reaction observed under EDTA conditions.

At interface 2, charge neutralization and charge reversal of residue D202 of the N-core domain both afforded ~6-fold stimulation of the reaction and, as expected from previous results, reduced the calcium dependence of telomere resolution (Fig 2B; see also [16]). The C-terminal helix terminates in RKG residues that may be in place for interactions with D202. We generated a deletion mutant that truncated TelA to delete these last 3 residues (ΔC3) and found this mutant to provide 8.1-fold reaction stimulation relative to wild type TelA, with Ca²⁺. Charge neutralization or charge reversal of both the R440 and K441 residues in tandem produced 4.2-fold and 11.2-fold stimulation with Ca²⁺, respectively, in the resulting double mutants.

Because the mutations we generated at the hypothesized autoinhibitory interfaces were found to be mostly stimulatory there was less concern that the mutations had led to their phenotypes by inducing misfolding of the resulting proteins. Nonetheless, we assessed the effect of the mutations on other activities of TelA to control for such potential effects. TelA has been characterized to be a robust single-stranded DNA annealing protein and it is known that a properly folded, full-length TelA is required for optimal annealing [13]. Therefore, we assayed our mutants with a stringent annealing assay employing the structured HIV transactivational response element (HIV_TAR; S3 and S4 Figs in S1 File). Single mutants of the two interfaces showed no dramatic hypo- or hyperactivity in the annealing assays.

Combining activating mutations leads to additive activation of TelA

The characterization of TelA mutants that weakened the hypothesized interactions on one side of the two interfaces produced a range of activation from 2-18-fold. Therefore, it seemed plausible that both interfaces contributed to autoinhibition of TelA’s telomere resolution activity. If the two interfaces are independent of each other one would predict that the effect of combining activating mutations from the two interfaces would be additive (i.e. greater than the activation afforded by either mutation alone). If, instead, the mutations affected a common interface, combining mutations would not be expected to result in activation greater than the most activating single mutation. To test this prediction we generated double mutants that combined activating mutations from interface 1 and interface 2 (Fig 3).

Fig 3 — Graphical summary of the initial rates of telomere resolution conducted at 0°C with 1 mM EDTA vs. 2 mM CaCl₂. Shown is the mean and standard deviation of 3 independent replicates excepting the ΔC3; D202AE337K and ΔC3; D202RE337K mutants that were assayed 6 times. ΔC3 denotes the deletion of the last 3 amino acids residues from the C-terminal domain. This removes R440 and K441 as well as the terminal glycine of TelA. The fold stimulation over wild type is shown.

We first tested the effect of combining the ΔC3 and D202A or D202R mutations. Since the interface 2 model posits these residues to be interacting to help form the interface we were surprised to find that the activation afforded by the ΔC3 and D202A or D202R mutations to be mildly additive. This may reflect the role of D202 in reaction responsiveness to divalent metal ions [16]. Therefore, in an effort to obtain optimally activated mutants both (ΔC3; D202A) and (ΔC3; D202R) were combined with the optimally activating interface 1 mutation E337K.

The combination of the interface 1 (E337K) mutation with either of the interface 2 mutations (ΔC3; D202A and ΔC3; D202R) resulted in TelA variants that exhibited additive stimulation (55-124-fold stimulation). The (ΔC3; D202RE337K) mutant, in particular, shows greater than 89-fold stimulation of the initial rate in EDTA conditions and a 124-fold stimulation, over wild type TelA, in Ca²⁺ buffer conditions. The combination of activating mutations from the two hypothesized interfaces with mutations that, simultaneously, reduce the divalent metal ion dependence of telomere resolution has produced a largely metal insensitive and hyperactive mutant (Fig 3B). As expected, the tested mutants were found to have approximately wild type levels of annealing proficiency indicating no gross misfolding issues (S4 Fig in S1 File).

Testing the modeled autoinhibitory interface interactions with paired charge reversal mutagenesis

If the details of the modeled autoinhibitory interfaces in TelA are accurate, one would predict that combining charge reversal mutations on both sides of the interfaces would result in suppression of the activation observed with single charge reversal mutants by reinstating electrostatic interactions at the proposed interfaces. To test the possible charge-charge interactions at interface 1 we combined charge reversal mutations R6E, K7E and K9E with either D333K or E337K (Fig 4). In both cases there was a pattern of activity where R6E had the least effect on activity, K7E had an intermediate effect and K9E reduced activity the most when combined with either D333K or E337K. This suggested that K7E and K9E interacted with D333K and E337K. However, examination of the double mutants in the annealing assay revealed hypoactivity in this assay for both the (K9ED333K) and (K9EE337K) mutants, indicating that these proteins are likely improperly folded (S4 Fig in S1 File). The (K9E), (D333K) and (E337K) single mutants were wild type in the annealing assay (S3 Fig in S1 File). In aggregate, these results provide some evidence that the K7 residue may interact with D333 and/or E337 in TelA to establish autoinhibitory interface 1.

Fig 4 — A) Graphical summary of the initial rates of telomere resolution conducted at 0°C with 1 mM EDTA vs. 2 mM CaCl₂ of interface 1 charge reversal single and double mutants. Shown is the mean and standard deviation of at least 3 independent replicates. B) Graphical summary of the initial rates of telomere resolution conducted at 0°C with 1 mM EDTA vs. 2 mM CaCl₂ of interface 2 charge reversal mutants. Shown is the mean and standard deviation of 3 independent replicates, excepting for the R6EE337K and K9EE337K mutants that had 6 replicates. RK-2E denotes the TelA double charge reversal mutations R440EK441E. The fold stimulation over wild type is shown.

To test possible interactions at interface 2 we combined the D202R mutation with the dual charge reversal mutations of the terminal RK motif at the end of TelA (R440EK441E). The (R440EK441E) mutant showed a robust 11.2-fold activation over wild type TelA. The combination of the D202R mutation with R440EK441E, unexpectedly, resulted in further activation of TelA (88.4-fold with EDTA and 61.6-fold activation with Ca²⁺ reaction conditions; Fig 4). This combination of mutations has eliminated the divalent metal-dependence of the reaction. The (D202RR440EK441E) triple mutant was found to have wild type proficiency in annealing assays (S5 Fig in S1 File). These results do not support the hypothesis that D202 interacts with R440 and/or K441 to establish autoinhibitory interface 2.

Testing the modeled autoinhibitory interface 1 interactions with deletion analysis and N-terminal domain add back experiments

The use of paired charge reversal mutations across the putative autoinhibitory interfaces provided modest evidence of potentially interacting residues at interface 1. In order to provide a more stringent test of the proposed interactions we assessed the effect of the most stimulatory interface 1 point mutation (E337K) in a background where the interacting N-terminal domain has been deleted (ΔN; E337K). Measuring the initial rate at 0°C revealed that the ΔN mutant was unreactive at this reaction temperature. However, the double mutant (ΔN; E337K) showed the greatest degree of reaction activation seen yet with any of our mutants (>200-fold in both EDTA and Ca²⁺ conditions; Fig 5B). This strongly suggested that the E337K mutation must activate the reaction, at least in part, by mechanisms independent of a contribution to the hypothesized autoinhibitory interface 1.

Fig 5 — A) Schematic summary of the domain structure of TelA followed by a schematic of the approach of combining the N-terminal domain deletion mutant of TelA (ΔN) with an excess of the N-terminal domain (N) added in *trans* to assess the inhibitory potential of the exogenously added N-terminal domain. Residue numbers delimiting the TelA domains is shown in the first graphic. B) Graphical summary of the initial rates of telomere resolution conducted at 0°C (left graph) or 30°C (right graph) using buffers containing 1 mM EDTA vs. 2 mM CaCl₂. Shown is the mean and standard deviation of 3 independent replicates excepting the ΔN; D202RE337K mutant that had 6 replicates. Where the N-terminal domain is added in *trans* (+N) it is added in 10.5-fold molar excess over the ΔN mutant. The fold stimulation over wild type is shown.

To reconfirm our previous study that adding the N-terminal domain, in trans, to reactions with the ΔN mutant leads to reaction inhibition we measured the initial rate of reaction of the (ΔN; E337K) mutant with a 10-fold molar excess of the N-terminal domain added, in trans ([16]; Fig 5B). Adding the N-terminal domain (N) reduced the reaction rate of (ΔN; E337K) ~22-fold, indicating that the N-terminal domain does have the ability to inhibit TelA activity. Testing the effect of adding the N-terminal domain harbouring the activating mutation K9E, in trans, (N; K9E) resulted in a lesser degree of reaction inhibition (~10-fold) compared to the ~22-fold reduction afforded by adding wild type N. We assessed the effect of adding (N) and (N; K9E) to reactions with (ΔN) by running assays at the standard reaction temperature of 30°C (Fig 5B). The same pattern of inhibition seen with the (ΔN; E337K) mutant was obtained with (ΔN), except that there was a greater dependence on inhibition of having Ca²⁺-free conditions employed, consistent with the effect of the E337K mutation lowering the Ca²⁺-dependence of telomere resolution (Fig 2).

The contradictory results from the activation afforded by combining ΔN with E337K to make a double mutant (activation) vs. combining ΔN and E337K in N-terminal domain add back experiments (inhibition) highlighted the possibility that the phenotype caused by the E337K and K9E mutations could be due to factors distinct from weakening the hypothesized interface 1. We examined (ΔN) and (ΔN; E337K) in annealing assays and confirmed previous results that the presence of the N-terminal domain is necessary for annealing reactions (S5B Fig in S1 File; [16]). Using the bulged hairpin structured HIV_TAR element we also found the N-terminal domain on its own to be a poor annealing protein when provided with substrates with strong secondary structure. The N-terminal domain with the K9E mutation (N; K9E) showed even worse annealing activity than N (S5B Fig in S1 File). We have never been able to detect bandshifts of substrate DNA by (ΔN) in electromobility shift assay (EMSA) experiments and this continued to be the case in this study. However, we did detect bandshifts using (ΔN; E337K). S6B, S6C Fig in S1 File documents EMSA experiments with (ΔN) and (ΔN; E337K) using substrate replicated telomeres (rTels) and hp products. We infer from the pattern of binding activity that (ΔN; E337K) resolves the rTel and that it is the hp product that (ΔN; E337K) was binding to produce a bandshift in our assays. (ΔN) was unable to bandshift either rTel or hp product (S6B, S6C Fig in S1 File). We also examined the N-terminal domain (N) and (N; K9E) in EMSA experiments to determine if the K9E mutation had affected the non-specific DNA binding activity of the N-terminal domain (S6D Fig in S1 File). (N) shows a laddering bandshift indicative of successively more N loading onto the rTel substrate. However, (N; K9E) failed to bind DNA; this is consistent with the abrogated annealing activity seen in S5B Fig in S1 File. We had established in a previous study that interaction of (N) with (ΔN) in crosslinking studies was promoted by the presence of DNA [16]. It seems likely that the lesser degree of reaction inhibition seen with (N; K9E) added, in trans, to (ΔN) and (ΔN; E337K) is due to this loss of DNA binding activity caused by the K9E mutation. Finally, we assessed the behaviour of another of our hyperactive TelA mutants (ΔC3; D202RE337K) in an EMSA experiment with an rTel vs. wild type TelA (S6D Fig in S1 File). Wild type TelA produced c1 and c2 complexes inferred to be the binding of one protomer (c1) and two protomers of TelA (c2) while the hyperactive (ΔC3; D202RE337K) mutant again resolved the rTel and bandshifted the hp product (c3). This indicates that the binding behavior of the (ΔN; E337K) is a general feature of activation by the E337K mutation rather than a feature specific to the ΔN background.

Finally, we expected that combining our most activating mutations together would produce optimally activated TelA variants. We tested this by examining (ΔN; D202RE337K), (ΔNΔC3; E337K) and (ΔNΔC3; D202RE337K) mutants for reaction stimulation (Fig 5B). Both (ΔN; D202RE337K) and (ΔNΔC3; D202RE337K) were highly activated for both EDTA and Ca²⁺ conditions but (ΔNΔC3; E337K) was poorly active. This unexpectedly poor activity is likely due to a folding issue as it is the only protein of the three tested that had defects in annealing and substrate binding (S7 Fig in S1 File).

Does hyperactivation of TelA unleash competing or inappropriate activities?

We assessed the effect of hyperactivation of TelA on competing activities with a collection of mutants that showed >50-fold activation of the initial rates of reaction. First we wanted to test these mutants on a negatively supercoiled substrate plasmid since it is known to be a poor substrate for resolution; resolution normally requires either relaxation of the supercoils or linearization of the substrate DNA (Fig 6A; [6, 11]). Wild type TelA and most of the mutants showed little activity in this assay. The highly activated (ΔN; D202RE337K) and (ΔNΔC3; D202RE337K) mutants were, unexpectedly, able to resolve the supercoiled substrate. The gel shows a ladder of topoisomers indicating that these mutants may have relaxed the plasmid enough to be able to resolve it.

We next tested if the hyperactive mutants tried to resolve a mutant rTel that has been asymmetrized between the scissile phosphates; thereby inhibiting hairpin formation. None of the hyperactivated mutants showed inappropriate activity on this substrate (that would produce TelA blocked double-strand breaks). Reaction with a negatively supercoiled version of this plasmid substrate revealed topoisomerase activity for each mutant on this substrate; indicating that they are all active but locked into abortive DNA cleavage and rejoining reactions (S8 Fig in S1 File).

We also tested if the competing side reaction of site-specific recombination between rTels was unmasked by the activating mutations (Fig 6C). Wild type TelA is prohibited from promoting this reaction, in part, by autoinhibition by the N-terminal domain [16]. The (ΔC3; D202AE337K) and (ΔN; E337K) mutants were also inactive for recombination but the remaining mutants were all able to form Holliday junctions. This pattern of reactivity suggests that the D202 residue may also play a role in masking this latent activity and that reversal of the charge at this position can also activate this activity (Fig 6C). It is unclear why combining the E337K mutation with the N-terminal domain deletion has suppressed the recombination that we know the ΔN mutation allows [16].

Finally, we assessed whether the activating mutations in the hyperactive TelAs had unleashed the latent ability of the enzyme to promote the reverse reaction of hp telomere fusion [16, 17]. Fig 7A shows an analysis with telomeric suicide half-sites to assess the DNA cleavage activity of wild type TelA and the hyperactive TelA mutants. The incubation was at 5°C overnight, a condition that favours the reverse reaction. All proteins showed a relatively uniform level of DNA cleavage activity with the telomeric half-site (32–40%). In the assay with the hp telomere our positive control for telomere fusion was provided by the reaction of the (ΔN) mutant supplemented with excess N-domain provided in trans (59% conversion; [16]). Most of our hyperactivated mutants were also activated for reaction reversal without the need for adding excess N-terminal domain (18.4–38.5% conversion). Wild type TelA and the (ΔC3; D202AE337K) and the (ΔN; E337K) mutants were the exception; being hyperactivated for telomere resolution without unmasking the latent telomere fusion reaction (Fig 7B). All TelA variants were inactive when tested with mock telomeric half-site and hairpin substrates, confirming the site-specificity of all the hyperactivated versions of TelA (Fig 7A & 7B).

Fig 7 — A) Half-site cleavage assay with hyperactive TelA mutants. On the left is a graphic of the cleavage reaction in which TelA becomes covalently trapped on a suicide half-site after DNA cleavage. On the right are 8%PAGE 1X TAE/0.1% SDS gel panels run with 150 nM of the indicated hyperactive TelA mutants incubated at 5°C for 17 h. The numbers below the lanes in the telomeric half-site panel reflect percent DNA cleavage of the half-site. hs denotes halfsite; CP denotes cleavage products. The difference in mobility of the cleavage products is due to the smaller size of the ΔN mutants. B) Telomere fusion assays with hyperactive TelA mutants. On the left is a graphic of the hp telomere fusion reaction with a hp telomere producing an *rTel* junction (reaction reversal). On the right are 8%PAGE 1X TAE/0.1% SDS gel panels run with 150 nM of the indicated hyperactive TelA mutants at 5°C for 17 h. The numbers below the lanes in the hp telomere panel reflect percent conversion from hp telomeres to fused *rTel* junctions. hp denotes hairpin telomere; *rTel* denotes a replicated telomere junction formed by fusing hp telomeres; CP denotes cleavage products.

Testing the most useful hyperactive mutants at standard reaction temperature

Our analysis from Figs 6 & 7 indicated that the (ΔC3; D202AE337K) and (ΔN; E337K) mutants were successfully hyperactivated without unleashing inappropriate competing reactions. The telomere resolution activity of all our mutants was assessed at the unusual reaction temperature of 0°C to facilitate easy comparison of the initial rates for proteins spanning a wide range of rates (Figs 2–5). We repeated the telomere resolution analysis of this pair of utile hyperactive mutants and compared their activation relative to wild type TelA in reactions conducted at the standard reaction temperature of 30°C. In order to slow down the reaction sufficiently to measure rates we reduced the TelA concentration in the assay from the 74 nM concentration used at 0°C to 3 nM for assays conducted at 30°C (Fig 8). Wild type TelA did not have detectable activity in EDTA conditions at this low TelA concentration but had a measurable rate with Ca²⁺ containing conditions. Both hyperactive mutants showed robust hyperactivation at 30°C indicating that the hyperactivity of these mutants was not, primarily, due to a relief of the cold-sensitivity of the telomere resolution reaction. It is noteworthy that differential sensitivity to cold reaction temperatures does play some role in the observed reaction stimulation as the (ΔC3; D202AE337K) mutant is more active than the (ΔN; E337K) mutant at 30°C whereas the opposite was true at 0°C.

Fig 8 — Graphical summary of the initial rates of telomere resolution conducted at 30°C using 3 nM TelA showing the rates of the reaction incubated without divalent metal ions (1 mM EDTA) compared to the rates of reaction in the presence of 2 mM CaCl₂. Shown is the mean and standard deviation of 3 independent replicates. The values over the individual bars represent the fold stimulation over the equivalent conditions for wild type TelA. nc denotes not calculated since the fold stimulation for the EDTA conditions were not calculated due to the lack of measurable activity for wild type TelA in EDTA.

A summary of the assay results for all tested mutants is presented in Table 1.

Table 1. Summary of mutant phenotypes.

Protein	tel. res. ¹	hp fusion	recombin.	topo.	annealing	rTel bind
wild type	1	no	no	no	normal	normal
K5D	2.7	N/T	N/T	N/T	normal	N/T
R6E	2.3	N/T	N/T	N/T	normal	N/T
K7E	1.4	N/T	N/T	N/T	normal	N/T
K9A	7.7	N/T	N/T	N/T	normal	N/T
K9E	3.9	N/T	N/T	N/T	normal	N/T
D332A	0.8	N/T	N/T	N/T	normal	N/T
D333A	7.2	N/T	N/T	N/T	normal	N/T
D333K	2.6	N/T	N/T	N/T	normal	N/T
E337A	11.6	N/T	N/T	N/T	normal	N/T
E337K	18.5	N/T	N/T	N/T	normal	N/T
D202A	6.5	N/T	N/T	N/T	normal	N/T
D202R	6.8	N/T	N/T	N/T	normal	N/T
R440AK441A (RK-2A)	4.2	N/T	N/T	N/T	normal	N/T
R440EK441E (RK-2E)	11.2	N/T	N/T	N/T	normal	N/T
R6ED333K	2.6	N/T	N/T	N/T	very poor	N/T
K7ED333K	1.7	N/T	N/T	N/T	normal	N/T
K9ED333K	0.1	N/T	N/T	N/T	very poor	N/T
R6EE337K	16.4	N/T	N/T	N/T	normal	N/T
K7EE337K	12.8	N/T	N/T	N/T	normal	N/T
K9EE337K	6.4	N/T	N/T	N/T	very poor	N/T
D202R RK-2E	61.6	yes	yes	no	normal	N/T
TelA (1–439) ΔC3	8.1	N/T	N/T	N/T	normal	N/T
ΔC3; D202A	14.2	N/T	N/T	N/T	normal	N/T
ΔC3; D202AE337K	84.2	no	no	no	normal	normal
ΔC3; D202RE337K	89.4	yes	yes	no	normal	normal
ΔN	0	no	yes	no	no	no
ΔN; E337K	218	no	no	no	no	normal
ΔN; D202RE337K	156	yes	yes	yes	no
ΔNΔC3; E337K	0.8	N/T	N/T	N/T	very poor	very poor
ΔNΔC3; D202RE337K	198	yes	yes	yes	normal	normal
N	no	N/T	N/T	N/T	poor	normal
N; K9E	no	N/T	N/T	N/T	very poor	no

Open in a new tab

tel. res. denotes telomere resolution; recombin. denotes recombination; topo. denotes topoisomerase activity on a wild type substrate plasmid; rTel bind denotes EMSA assays on substrate DNA; N/T denotes not tested. Bolded text within the cells indicates hypoactive activities indicative misfolded proteins. Italicized text within the cells indicates these inappropriate activities have been unleashed due loss of autoinhibition.

¹ fold-stimulation over wild type TelA measured at 0°C in the presence of 2 mM CaCl₂.

Discussion

In the present study we have interrogated an AlphaFold2 model of a TelA monomer unbound to DNA that predicted the presence of two autoinhibitory interfaces. The predicted aligned error plot for the model is presented in S2 Fig in S1 File and indicates a low confidence (~30 Å) in the prediction for interface 1 but much higher confidence for interface 2 (~10 Å). We interrogated the two modeled autoinhibitory interfaces by generating and purifying TelA mutants predicted to weaken and/or abolish these interfaces and assessing their telomere resolution activity. The results called to mind the famous aphorism coined by the statistician George Box that “All models are wrong, but some models are useful” [24]. While our mutagenic interrogation of the modeled interfaces did not support the details of the model, the model was, nonetheless, useful in discovering activating mutations that, when combined, led to a dramatic (50-280-fold) activation of telomere resolution by TelA. For most hyperactive mutants the resulting activation has come at the expense of activating competing reactions that telomere resolvases can promote. Vitally, we have also recovered hyperactive mutants that retain enough autoinhibition not to promote the competing reactions of hp telomere fusion or recombination between rTel junctions.

Possession of enzymes that promote DNA breakage and rejoining activities is risky as unrepaired strand scissions can lead to genomic instability and cell death. Unsurprisingly, such enzymes are under multiple levels of control/regulation [25–27]. The most fundamental control mechanisms are intrinsic to the enzyme itself, often in the form of autoinhibition [28]. An example from an enzyme class structurally and mechanistically related to the telomere resolvases is the tyrosine recombinase Cre. Cre promotes recombination between short inverted repeat recognition sites called loxP; Cre synapses a pair of loxP sites together via assembly of a tetramer of Cre [29]. The tetrameric assembly possesses two-fold symmetry that helps define active protomers vs. inactive protomers, as Cre executes a pairwise series of DNA cleavage and strand transfer steps, transiting through a HJ intermediate to produce recombinants [30–32]. A key aspect of this two-fold symmetry is the trans swapping of the C-terminal helix of Cre between protomers. Cre has been observed in a monomeric state before DNA binding in which the C-terminal helix is stacked in cis over the DNA binding cleft and active site of unbound Cre. DNA binding induces a cis-to-trans switch in the conformation of the C-terminal helix [33]. Our modeling suggested that TelA may also undergo such a cis-to-trans switch in the conformation of the C-terminal helix between the transition from unbound monomer to DNA bound dimer (Fig 1 and S2 Fig in S1 File). While the model details we tested were not supported by the mutagenic study of the charged residues D202, R440 and K441 the strong stimulation of the reaction by mutants of these residues lends some support to this hypothesis. The specific reason mutation of these residues was activating may have been confounded by the role the D202 residue plays in divalent metal ion responsiveness [16]. Interestingly, the D202R mutation could also be stimulatory due to enhanced DNA binding as the charge reversal at this position is predicted to allow additional DNA contacts with the hairpin turnaround in the crystallographic model with DNA (S9 Fig in S1 File). We were hampered in our ability to test other residues at this interface in positions modeled with higher confidence (<10Å) by the high density of residues in the hairpin binding module important in activity for telomere resolution [12].

A further, much lower confidence (~30Å), autoinhibitory interaction is hypothesized between the N-terminal domain and the catalytic domain (Fig 1, S2 Fig in S1 File and ref. [16]). Our paired charge reversal analysis lent some support for K7 interactions with D333 and potentially with E337 (Fig 4). However, the E337K mutation increases the affinity of TelA for the substrate DNA and, thus, affords stimulation by means independent of a contribution to an autoinhibitory interface (S6 Fig in S1 File). We conclude this because deletion of the N-terminal domain plus incorporation of the E337K mutation that increases affinity for rTel DNA led to additional TelA activation rather than the expected neutral effect. Addition of the N-terminal domain in trans inhibited telomere resolution confirming the autoinhibitory effect of the N-terminal domain but indicating that multiple or alternative interfaces likely exist beyond the one modeled. The D333K and E337K mutations were introduced digitally into a crystallographic model of TelA complexed with DNA; increased DNA affinity is not predicted by this analysis without some conformational change in TelA as the residues point away from the substrate DNA (S9 Fig in S1 File).

The (ΔN; E337K) mutant has fulfilled the principle prediction of the N-terminal domain’s hypothesized role as an autoinhibitory domain of deletion of the domain leading to hyperactivity of the enzyme [28]. The ΔN mutant was activated for competing reactions under certain conditions but did not show hyperactivity in telomere resolution (ref. [16] and Fig 5B). The lack of hyperactivity was due to the loss of affinity with the rTel substrate DNA. However, incorporation of the E337K mutation has reversed this loss of DNA affinity and revealed the activation afforded by loss of the N-terminal domain (>200-fold activation for EDTA and Ca²⁺ conditions). The (ΔN; E337K) mutant will have particularly high utility as an activated version of TelA since it retains a sufficient degree of autoinhibition, presumably by cis interactions of the C-terminal helix, not to be activated for the competing reactions of hp telomere fusion and recombination between rTels (Figs 6 & 7). Also of great potential utility is the (ΔC3; D202AE337K) mutant that has hyperactivated TelA without activating competing reactions, presumably by continued autoinhibition via the N-terminal domain. Our collection of hyperactive TelA mutants will also be useful for future studies, perhaps by cryo-EM, to characterize the structural basis of autoinhibition and activation of TelA.

Supporting information

S1 Raw images

(PDF)

pone.0307590.s001.pdf^{(1.1MB, pdf)}

S1 File

(PDF)

pone.0307590.s002.pdf^{(1.3MB, pdf)}

Acknowledgments

We would like to thank lab and research cluster members for fruitful discussions.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

KK was supported by Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN 04382-2017, GRF-0-2006, RGPIN-2024-05101) and by a CoMRAD grant from the University of Saskatchewan's College of Medicine (2022-2023). There was no additional external funding received for this study.

References

1.Picardeau M, Lobry JR, Hinnebusch BJ. Physical mapping of an origin of bidirectional replication at the centre of the Borrelia burgdorferi linear chromosome. Mol Microbiol. 1999;32(2):437–45. [DOI] [PubMed] [Google Scholar]
2.Picardeau M, Lobry JR, Hinnebusch BJ. Analyzing DNA strand compositional asymmetry to identify candidate replication origins of Borrelia burgdorferi linear and circular plasmids. Genome Res. 2000;10(10):1594–604. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Ravin NV, Kuprianov VV, Gilcrease EB, Casjens SR. Bidirectional replication from an internal ori site of the linear N15 plasmid prophage. Nucleic Acids Res. 2003;31(22):6552–60. doi: 10.1093/nar/gkg856 . [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Chaconas G, Stewart PE, Tilly K, Bono JL, Rosa P. Telomere resolution in the Lyme disease spirochete. EMBO J. 2001;20(12):3229–37. doi: 10.1093/emboj/20.12.3229 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Kobryn K, Chaconas G. The circle is broken: Telomere resolution in linear replicons. Current Opinion in Microbiology. 2001;4:558–64. doi: 10.1016/s1369-5274(00)00251-4 [DOI] [PubMed] [Google Scholar]
6.Kobryn K, Chaconas G. ResT, a telomere resolvase encoded by the Lyme disease spirochete. Mol Cell. 2002;9(1):195–201. doi: 10.1016/s1097-2765(01)00433-6 . [DOI] [PubMed] [Google Scholar]
7.Deneke J, Ziegelin G, Lurz R, Lanka E. The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity. Proc Natl Acad Sci U S A. 2000;97(14):7721–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Aihara H, Huang WM, Ellenberger T. An interlocked dimer of the protelomerase TelK distorts DNA structure for the formation of hairpin telomeres. Mol Cell. 2007;27(6):901–13. doi: 10.1016/j.molcel.2007.07.026 . [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Shi K, Huang WM, Aihara H. An enzyme-catalyzed multistep DNA refolding mechanism in hairpin telomere formation. PLoS Biol. 2013;11(1):e1001472. doi: 10.1371/journal.pbio.1001472 . [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Kobryn K, Chaconas G. Hairpin telomere resolvases. Microbiol Spectrum. 22014. p. MDNA-0023-2014. [DOI] [PubMed] [Google Scholar]
11.Huang WM, DaGloria J, Fox H, Ruan Q, Tillou J, Shi K, et al. Linear chromosome-generating system of Agrobacterium tumefaciens C58: protelomerase generates and protects hairpin ends. J Biol Chem. 2012;287(30):25551–63. doi: 10.1074/jbc.M112.369488 . [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Balouchi M, Huang SH, McGrath SL, Kobryn K. The telomere resolvase, TelA, utilizes an underwound pre-cleavage intermediate to promote hairpin telomere formation. PLoS One. 2023;18(11):e0294732. Epub 20231129. doi: 10.1371/journal.pone.0294732 ; PubMed Central PMCID: PMC10686437. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.McGrath SL, Huang S. H. Kerri Kobryn. Single stranded DNA annealing is a conserved activity of telomere resolvases. PLoSOne. 2021;16(2):e0246212. doi: 10.1371/journal.pone.0246212 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Mir T, Huang SH, Kobryn K. The telomere resolvase of the Lyme disease spirochete, Borrelia burgdorferi, promotes DNA single-strand annealing and strand exchange. Nucleic Acids Res. 2013;41(22):10438–48. doi: 10.1093/nar/gkt832 . [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Huang SH, Kobryn K. The Borrelia burgdorferi telomere resolvase, ResT, anneals ssDNA complexed with its cognate ssDNA-binding protein. Nucleic Acids Res. 2016;44(11):5288–98. Epub 2016/05/01. gkw344 [pii] doi: 10.1093/nar/gkw344 ; PubMed Central PMCID: PMC4914115. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.McGrath SL, Huang SH, Kobryn K. The N-terminal domain of the Agrobacterium tumefaciens telomere resolvase, TelA, regulates its DNA cleavage and rejoining activities. J Biol Chem. 2022;298(5):101951–64. doi: 10.1016/j.jbc.2022.101951 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Kobryn K, Chaconas G. Fusion of hairpin telomeres by the B. burgdorferi telomere resolvase ResT: Implications for shaping a genome in flux. Mol Cell. 2005;17(6):783–91. doi: 10.1016/j.molcel.2005.02.025 . [DOI] [PubMed] [Google Scholar]
18.Kobryn K, Briffotaux J, Karpov V. Holliday junction formation by the Borrelia burgdorferi telomere resolvase, ResT: implications for the origin of genome linearity. Mol Microbiol. 2009;71(5):1117–30. Epub 2009/01/28. MMI6584 [pii] doi: 10.1111/j.1365-2958.2008.06584.x . [DOI] [PubMed] [Google Scholar]
19.Bankhead T, Kobryn K, Chaconas G. Unexpected twist: harnessing the energy in positive supercoils to control telomere resolution. Mol Microbiol. 2006;62:895–905. doi: 10.1111/j.1365-2958.2006.05423.x . [DOI] [PubMed] [Google Scholar]
20.Tourand Y, Lee L, Chaconas G. Telomere resolution by Borrelia burgdorferi ResT through the collaborative efforts of tethered DNA binding domains. Mol Microbiol. 2007;64(3):580–90. doi: 10.1111/j.1365-2958.2007.05691.x . [DOI] [PubMed] [Google Scholar]
21.Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583–9. Epub 20210715. doi: 10.1038/s41586-021-03819-2 ; PubMed Central PMCID: PMC8371605. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Bankhead T, Chaconas G. Mixing active site components: A recipe for the unique enzymatic activity of a telomere resolvase. Proc Natl Acad Sci USA. 2004;101(38):13768–73. doi: 10.1073/pnas.0405762101 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Lucyshyn D, Huang SH, Kobryn K. Spring loading a pre-cleavage intermediate for hairpin telomere formation. Nucleic Acids Res. 2015;43(12):6062–74. Epub 2015/05/27. gkv497 [pii] doi: 10.1093/nar/gkv497 . [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Box GEP, Draper NR. Empirical model-building and response surfaces. New York; Chichester: Wiley; 1987. xiv,669p: ill p. [Google Scholar]
25.Grundy GJ, Yang W, Gellert M. Autoinhibition of DNA cleavage mediated by RAG1 and RAG2 is overcome by an epigenetic signal in V(D)J recombination. Proc Natl Acad Sci U S A. 2010;107(52):22487–92. Epub 20101213. doi: 10.1073/pnas.1014958107 ; PubMed Central PMCID: PMC3012499. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Lu C, Ward A, Bettridge J, Liu Y and Desiderio S. An Autoregulatory Mechanism Imposes Allosteric Control on the V(D)J Recombinase by Histone H3 Methylation. Cell Reports. 2015;10:29–38. doi: 10.1016/j.celrep.2014.12.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Jones M, Beuron F, Borg A, Nans A, Earl CP, Briggs DC, et al. Cryo-EM structures of the XPF-ERCC1 endonuclease reveal how DNA-junction engagement disrupts an auto-inhibited conformation. Nat Commun. 2020;11(1):1120. Epub 20200228. doi: 10.1038/s41467-020-14856-2 ; PubMed Central PMCID: PMC7048804. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Pufall MA, Graves BJ. Autoinhibitory domains: modular effectors of cellular regulation. Annu Rev Cell Dev Biol. 2002;18:421–62. Epub 20020402. doi: 10.1146/annurev.cellbio.18.031502.133614 . [DOI] [PubMed] [Google Scholar]
29.Gopaul DN, Duyne GD. Structure and mechanism in site-specific recombination. Curr Opin Struct Biol. 1999;9(1):14–20. doi: 10.1016/s0959-440x(99)80003-7 [DOI] [PubMed] [Google Scholar]
30.Guo F, Gopaul DN, van Duyne GD. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature. 1997;389(6646):40–6. doi: 10.1038/37925 . [DOI] [PubMed] [Google Scholar]
31.Gopaul DN, Guo F, Van Duyne GD. Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. Embo J. 1998;17(14):4175–87. doi: 10.1093/emboj/17.14.4175 . [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Ghosh K, Lau CK, Gupta K, Van Duyne GD. Preferential synapsis of loxP sites drives ordered strand exchange in Cre-loxP site-specific recombination. Nat Chem Biol. 2005;1(5):275–82. Epub 2006/01/13. nchembio733 [pii] doi: 10.1038/nchembio733 . [DOI] [PubMed] [Google Scholar]
33.Unnikrishnan A, Amero C, Yadav DK, Stachowski K, Potter D, Foster MP. DNA binding induces a cis-to-trans switch in Cre recombinase to enable intasome assembly. Proc Natl Acad Sci U S A. 2020;117(40):24849–58. Epub 20200923. doi: 10.1073/pnas.2011448117 ; PubMed Central PMCID: PMC7547242. [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0307590.r001

Decision Letter 0

Arthur J Lustig

2 Jun 2024

PONE-D-24-17544Design and Characterization of Hyperactive Mutants of the Agrobacterium tumefaciens Telomere Resolvase, TelA.PLOS ONE

Dear Dr. Kobryn,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Based on the critiques of reviewers and of my independent reading, the following comments need to be addressed:

I concur with Reviewer 1 that a score be presented the best fit AlphaFold model. A poor model might explain the lack of data support. This does not take of course take away from the interesting effects of the mutants just the logical framework of the data presentation.
Reviewer 1 also suggests testing in a subset of mutants, in which activity is varied by altering the concentration of the enzyme. Either conduct such an experiment for the most hyperactive mutant or provide a rebuttal as to why you feel that this is uncessary.
Answer all other critiques of both Reviewers.

Based on my reading of the text, I suggest the following additional changes to improve the clarity of the paper.

Page 12, line 9: It is unclear to me whether these events behave in a strictly additive way. To Please provide the expectations of additivity vs the results obtained within the text.
Page 13, Line 4. You say that D202 and deltaC-3 are additive. Please explain this outcome. Was this outcome expected?
Phenotypic separation: Since you discuss annealing activity and other enzyme characteristics at multiple points within the text, it would be very helpful to provide a summary table of all of the characteristics of each mutant/activity.
Redefine hp and rTel when first used in the Results section for clarity.
Figure 4, how many replicates of each mutant ws used to derive the mean and standard deviation.

Please submit your revised manuscript by Jul 17 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Arthur J. Lustig, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating in your Funding Statement:

"KK was supported by Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN 04382-2017, GRF-0-2006, RGPIN-2024-05101) and by a CoMRAD grant from the University of Saskatchewan's College of Medicine (2022-2023)."

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Huang et al. report the design and characterization of many mutant variants of Agrobacterium tumefaciens Telomere Resolvase, TelA. Despite the authors’ initial intention to relieve autoinhibition suggested by an AI-based structural model of DNA-free TelA, they identified several mutations that confer interesting gain-of-function phenotypes through independent and unknown mechanisms. The obtained mutant collection and biochemical data will be instrumental in further investigating the molecular mechanisms of hairpin telomere resolution or adapting TelA for biotechnology applications. Thus, the work seems to be well-suited for publication in this journal. I have several comments.

1. The AlphaFold prediction of DNA-unbound TelA served as the template for designing the mutants analyzed in this work. Although the individual domain structures are likely to be accurately predicted, it’s not clear if the domain-domain contacts were predicted with high confidence. The authors should show and discuss error estimates in the prediction, especially the Predicted Aligned Error. Also, the original paper reporting AlphaFold (PMID: 34265844) should be cited.

2. Most hairpin resolution assays were done at 0 deg, which is unusual. Data shown in Fig. 5B (WT vs. delta N) suggest that an enzyme completely inactive at 0 deg can be more active than WT at 30 deg. I don’t recommend repeating everything at 30 deg., but some of the most interesting mutants’ data (e.g., activities of the most active mutant) could be confirmed at 30 deg. It should be possible to slow the reaction by lowering the enzyme concentration, etc.

3. The authors should discuss the potential mechanisms of hyperactive mutants based on the DNA-bound TelA structure. For instance, are the D202R, D333K, and E337K substitutions predicted to generate additional DNA backbone contacts?

Reviewer #2: This manuscript is a mutagenic/biochemical analysis of reaction autoinhibition by the telomere resolvase (TelA) of Agrobacterium tumefaciens. The telomere resolvases are a unique class of DNA metabolizing enzyme that generate covalently closed hairpin ends on replication intermediates of linear replicons in several unrelated bacteria and phages. Although not abundantly found, these enzymes are fascinating as effective solutions for the end replication problem.

In the current work the authors begin with an AlphaFold model of TelA, including the N-terminal domain which is not resolved in the crystal structure and the absence of DNA which was present in the crystal structure. The model suggests two possible inhibitory interfaces which were then explored through alteration of putative participating residues coupled with in vitro analysis of the purified mutant proteins. The experiments are carefully performed and the data is judiciously interpreted. Although the results do not corroborate the molecular details of the model for the autoinhibitory interfaces, they do however, provide a variety of new information on autoinhibition and in particular residues involved in the process. The study has generated a variety of mutants with increased specific activity in telomere resolution activity, up to 280-fold improved over the wild-type enzyme, a very significant accomplishment. This work lays the ground work for some very interesting mechanistic characterization of the autoinhibition process in future by coupling the current data with future structural analysis of the entire TelA protein +/- DNA using cryo-EM; however, that is beyond the scope of the current work which makes a significant contribution to our knowledge of autoinhibition in TelA.

I have no concerns with the manuscript. The work is carefully executed and interpreted and the manuscript is well-constructed.

Minor point:

- Fig S7 – I can see the topoisomers, however the “topo” labelling mentioned in the legend is not on the figure.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Hideki Aihara

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2024 Jul 25;19(7):e0307590. doi: 10.1371/journal.pone.0307590.r002

Author response to Decision Letter 0

4 Jul 2024

Dear Dr. Lustig,

We thank you and the two reviewers for their expert evaluation of our manuscript ‘Design and Characterization of Hyperactive Mutants of the Agrobacterium tumefaciens Telomere Resolvase, TelA.’ (ONE-D-24-17544). We feel that addressing the reviewer comments/concerns has markedly strengthened the revised manuscript. In brief, we have added a Supplementary Figure detailing the predicted aligned error of the model used in the paper (S2 Fig). A second Supplementary Figure (S9 Fig) details the results of digital mutagenesis to assess introduction of polar contacts with D202R, D333K and E337K mutations with DNA potentially caused by these charge reversal mutations. We have also added an additional data figure in the main text of the paper (Figure 8) wherein we test the two most useful hyperactive mutants against wild type TelA at the standard reaction temperature of 30oC (instead of 0oC) using a low protein concentration to slow reactions for ease of measuring initial rates (3 nM as opposed to 74 nM). The mutants chosen for analysis were hyperactivated (>50-fold) but retained enough autoinhibition to suppress competing reactions. These changes have necessitated the renumbering of supplementary figure references and has lengthened, somewhat, the manuscript to incorporate discussion of this new figure and also discussion of the predicted aligned error results. What follows below is a point by point response to the reviewer/editor comments.

Editor comments:

Based on the critiques of reviewers and of my independent reading, the following comments need to be addressed:

1. I concur with Reviewer 1 that a score be presented the best fit AlphaFold model. A poor model might explain the lack of data support. This does not take of course take away from the interesting effects of the mutants just the logical framework of the data presentation.

We have prepared a new supplementary Figure with the Predicted Aligned Error (S2 Fig). This has necessitated the renumbering of the SFig referents throughout text. We have discussed the error estimates in the results and discussion sections. The take home is that the predicted aligned error estimates reveal that the model is low confidence for interface 1 and much higher confidence for interface 2. Nonetheless, testing both predicted interfaces proved a useful exercise for uncovering activating mutations.

3. Reviewer 1 also suggests testing in a subset of mutants, in which activity is varied by altering the concentration of the enzyme. Either conduct such an experiment for the most hyperactive mutant or provide a rebuttal as to why you feel that this is uncessary.

This was an excellent suggestion since the specific ranking of activities could be a read out of general activation but also, potentially, a read out of the effect of a mutation specifically on the cold-sensitivity of telomere resolution. We repeated telomere resolution assays for WT, (�C3; D202AE337K) and (�N; E337K) at 30oC at low protein concentration (3 nM). These two mutants were selected since they do not suffer from activation of inappropriate activities due to their hyperactivation. This analysis has become Fig. 8. The results indicate that activation is also seen at 30oC for these two mutants but the relative ranking of their activities has changed indicating that there were differential effects on the cold-sensitivity of the telomere resolution.

Based on my reading of the text, I suggest the following additional changes to improve the clarity of the paper.

Page 12, line 9: It is unclear to me whether these events behave in a strictly additive way. To Please provide the expectations of additivity vs the results obtained within the text.

What is meant by additive is now spelled out and a counter example of non-additive behaviour of a double mutant is provided if the mutations affect a common autoinhibitory interface. It essentially boils down to something akin to an in vitro epistasis analysis.

4. Page 13, Line 4. You say that D202 and deltaC-3 are additive. Please explain this outcome. Was this outcome expected?

We have clarified this section by indicating what our expectations when combining the D202 mutants with the delta-C3 mutation. The increased activation observed was unexpected and foreshadowed the result of the more rigorous paired charge reversal results documented later in the paper that these residues are unlikely to interact to help form the modeled interface.

5. Phenotypic separation: Since you discuss annealing activity and other enzyme characteristics at multiple points within the text, it would be very helpful to provide a summary table of all of the characteristics of each mutant/activity.

Such a mass of different assay results applied to numerous mutants has now been made more accessible, at a glance, by creation of Table 1 for the main body of the paper.

6. Redefine hp and rTel when first used in the Results section for clarity.

Completed as requested to improve clarity of presentation of the results.

7. Figure 4, how many replicates of each mutant ws used to derive the mean and standard deviation.

We have changed all figure legends to indicate that the graphed results show the mean and standard deviation of 3 independent replicates excepting the noted mutants that were assayed with 6 replicates. This affords greater clarity.

Reviewer #1 comments:

1) The AlphaFold prediction of DNA-unbound TelA served as the template for designing the mutants analyzed in this work. Although the individual domain structures are likely to be accurately predicted, it’s not clear if the domain-domain contacts were predicted with high confidence. The authors should show and discuss error estimates in the prediction, especially the Predicted Aligned Error.

Also, the original paper reporting AlphaFold (PMID: 34265844) should be cited.

The relevant Jumper et al. reference was in the results text but has also been added to the legend for Figure 1.

2) Most hairpin resolution assays were done at 0 deg, which is unusual. Data shown in Fig. 5B (WT vs. delta N) suggest that an enzyme completely inactive at 0 deg can be more active than WT at 30 deg. I don’t recommend repeating everything at 30 deg., but some of the most interesting mutants’ data (e.g., activities of the most active mutant) could be confirmed at 30 deg. It should be possible to slow the reaction by lowering the enzyme concentration, etc.

This was an excellent suggestion since the specific ranking of activities could be a read out of general activation but also, potentially, a read out of the effect of a mutation on the cold-sensitivity of telomere resolution. We repeated telomere resolution assays for WT, (�C3; D202AE337K) and (�N; E337K) at 30oC at low protein concentration (3 nM). These two mutants were selected since they do not suffer from activation of inappropriate activities due to their hyperactivation. This analysis has become Fig. 8. The results indicate that activation is also seen at 30oC for these two mutants but the relative ranking of their activities has changed indicating that there were differential effects on the cold-sensitivity of the telomere resolution.

3) The authors should discuss the potential mechanisms of hyperactive mutants based on the DNA-bound TelA structure. For instance, are the D202R, D333K, and E337K substitutions predicted to generate additional DNA backbone contacts?

We have performed and documented this ‘digital mutagenesis’ using an available TelA co-crystal structural model. This is now S9 Fig. The results show that D202R makes contacts with the DNA absent in the wild type but that D333K and E337K do not unless a modest conformational change occurs due to these mutations.

Reviewer #2 comments:

1) Fig S7 – I can see the topoisomers, however the “topo” labelling mentioned in the legend is not on the figure.

This supplementary figure is now S8 Fig and has the ladder of topoisomers bracketed and labeled as topo as noted in the figure legend. We thank the reviewer for catching this oversight.

We thank the reviewers for helping to strengthen the revised manuscript.

Best regards,

Kerri Kobryn, PhD

Associate Professor

Dept of Biochemistry, Microbiology & Immunology

College of Medicine

University of Saskatchewan

kerri.kobryn@usask.ca

Attachment

Submitted filename: Response to Reviewers.docx

pone.0307590.s003.docx^{(21.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0307590.r003

Decision Letter 1

Arthur J Lustig

9 Jul 2024

Design and Characterization of Hyperactive Mutants of the Agrobacterium tumefaciens Telomere Resolvase, TelA.

PONE-D-24-17544R1

Dear Dr. Kobryn,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Arthur J. Lustig, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Hideki Aihara

**********

PLoS One. doi: 10.1371/journal.pone.0307590.r004

Acceptance letter

Arthur J Lustig

16 Jul 2024

PONE-D-24-17544R1

PLOS ONE

Dear Dr. Kobryn,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Arthur J. Lustig

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Raw images

(PDF)

pone.0307590.s001.pdf^{(1.1MB, pdf)}

S1 File

(PDF)

pone.0307590.s002.pdf^{(1.3MB, pdf)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0307590.s003.docx^{(21.4KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0307590.ref001] 1.Picardeau M, Lobry JR, Hinnebusch BJ. Physical mapping of an origin of bidirectional replication at the centre of the Borrelia burgdorferi linear chromosome. Mol Microbiol. 1999;32(2):437–45. [DOI] [PubMed] [Google Scholar]

[pone.0307590.ref002] 2.Picardeau M, Lobry JR, Hinnebusch BJ. Analyzing DNA strand compositional asymmetry to identify candidate replication origins of Borrelia burgdorferi linear and circular plasmids. Genome Res. 2000;10(10):1594–604. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref003] 3.Ravin NV, Kuprianov VV, Gilcrease EB, Casjens SR. Bidirectional replication from an internal ori site of the linear N15 plasmid prophage. Nucleic Acids Res. 2003;31(22):6552–60. doi: 10.1093/nar/gkg856 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref004] 4.Chaconas G, Stewart PE, Tilly K, Bono JL, Rosa P. Telomere resolution in the Lyme disease spirochete. EMBO J. 2001;20(12):3229–37. doi: 10.1093/emboj/20.12.3229 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref005] 5.Kobryn K, Chaconas G. The circle is broken: Telomere resolution in linear replicons. Current Opinion in Microbiology. 2001;4:558–64. doi: 10.1016/s1369-5274(00)00251-4 [DOI] [PubMed] [Google Scholar]

[pone.0307590.ref006] 6.Kobryn K, Chaconas G. ResT, a telomere resolvase encoded by the Lyme disease spirochete. Mol Cell. 2002;9(1):195–201. doi: 10.1016/s1097-2765(01)00433-6 . [DOI] [PubMed] [Google Scholar]

[pone.0307590.ref007] 7.Deneke J, Ziegelin G, Lurz R, Lanka E. The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity. Proc Natl Acad Sci U S A. 2000;97(14):7721–6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref008] 8.Aihara H, Huang WM, Ellenberger T. An interlocked dimer of the protelomerase TelK distorts DNA structure for the formation of hairpin telomeres. Mol Cell. 2007;27(6):901–13. doi: 10.1016/j.molcel.2007.07.026 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref009] 9.Shi K, Huang WM, Aihara H. An enzyme-catalyzed multistep DNA refolding mechanism in hairpin telomere formation. PLoS Biol. 2013;11(1):e1001472. doi: 10.1371/journal.pbio.1001472 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref010] 10.Kobryn K, Chaconas G. Hairpin telomere resolvases. Microbiol Spectrum. 22014. p. MDNA-0023-2014. [DOI] [PubMed] [Google Scholar]

[pone.0307590.ref011] 11.Huang WM, DaGloria J, Fox H, Ruan Q, Tillou J, Shi K, et al. Linear chromosome-generating system of Agrobacterium tumefaciens C58: protelomerase generates and protects hairpin ends. J Biol Chem. 2012;287(30):25551–63. doi: 10.1074/jbc.M112.369488 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref012] 12.Balouchi M, Huang SH, McGrath SL, Kobryn K. The telomere resolvase, TelA, utilizes an underwound pre-cleavage intermediate to promote hairpin telomere formation. PLoS One. 2023;18(11):e0294732. Epub 20231129. doi: 10.1371/journal.pone.0294732 ; PubMed Central PMCID: PMC10686437. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref013] 13.McGrath SL, Huang S. H. Kerri Kobryn. Single stranded DNA annealing is a conserved activity of telomere resolvases. PLoSOne. 2021;16(2):e0246212. doi: 10.1371/journal.pone.0246212 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref014] 14.Mir T, Huang SH, Kobryn K. The telomere resolvase of the Lyme disease spirochete, Borrelia burgdorferi, promotes DNA single-strand annealing and strand exchange. Nucleic Acids Res. 2013;41(22):10438–48. doi: 10.1093/nar/gkt832 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref015] 15.Huang SH, Kobryn K. The Borrelia burgdorferi telomere resolvase, ResT, anneals ssDNA complexed with its cognate ssDNA-binding protein. Nucleic Acids Res. 2016;44(11):5288–98. Epub 2016/05/01. gkw344 [pii] doi: 10.1093/nar/gkw344 ; PubMed Central PMCID: PMC4914115. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref016] 16.McGrath SL, Huang SH, Kobryn K. The N-terminal domain of the Agrobacterium tumefaciens telomere resolvase, TelA, regulates its DNA cleavage and rejoining activities. J Biol Chem. 2022;298(5):101951–64. doi: 10.1016/j.jbc.2022.101951 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref017] 17.Kobryn K, Chaconas G. Fusion of hairpin telomeres by the B. burgdorferi telomere resolvase ResT: Implications for shaping a genome in flux. Mol Cell. 2005;17(6):783–91. doi: 10.1016/j.molcel.2005.02.025 . [DOI] [PubMed] [Google Scholar]

[pone.0307590.ref018] 18.Kobryn K, Briffotaux J, Karpov V. Holliday junction formation by the Borrelia burgdorferi telomere resolvase, ResT: implications for the origin of genome linearity. Mol Microbiol. 2009;71(5):1117–30. Epub 2009/01/28. MMI6584 [pii] doi: 10.1111/j.1365-2958.2008.06584.x . [DOI] [PubMed] [Google Scholar]

[pone.0307590.ref019] 19.Bankhead T, Kobryn K, Chaconas G. Unexpected twist: harnessing the energy in positive supercoils to control telomere resolution. Mol Microbiol. 2006;62:895–905. doi: 10.1111/j.1365-2958.2006.05423.x . [DOI] [PubMed] [Google Scholar]

[pone.0307590.ref020] 20.Tourand Y, Lee L, Chaconas G. Telomere resolution by Borrelia burgdorferi ResT through the collaborative efforts of tethered DNA binding domains. Mol Microbiol. 2007;64(3):580–90. doi: 10.1111/j.1365-2958.2007.05691.x . [DOI] [PubMed] [Google Scholar]

[pone.0307590.ref021] 21.Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583–9. Epub 20210715. doi: 10.1038/s41586-021-03819-2 ; PubMed Central PMCID: PMC8371605. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref022] 22.Bankhead T, Chaconas G. Mixing active site components: A recipe for the unique enzymatic activity of a telomere resolvase. Proc Natl Acad Sci USA. 2004;101(38):13768–73. doi: 10.1073/pnas.0405762101 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref023] 23.Lucyshyn D, Huang SH, Kobryn K. Spring loading a pre-cleavage intermediate for hairpin telomere formation. Nucleic Acids Res. 2015;43(12):6062–74. Epub 2015/05/27. gkv497 [pii] doi: 10.1093/nar/gkv497 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref024] 24.Box GEP, Draper NR. Empirical model-building and response surfaces. New York; Chichester: Wiley; 1987. xiv,669p: ill p. [Google Scholar]

[pone.0307590.ref025] 25.Grundy GJ, Yang W, Gellert M. Autoinhibition of DNA cleavage mediated by RAG1 and RAG2 is overcome by an epigenetic signal in V(D)J recombination. Proc Natl Acad Sci U S A. 2010;107(52):22487–92. Epub 20101213. doi: 10.1073/pnas.1014958107 ; PubMed Central PMCID: PMC3012499. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref026] 26.Lu C, Ward A, Bettridge J, Liu Y and Desiderio S. An Autoregulatory Mechanism Imposes Allosteric Control on the V(D)J Recombinase by Histone H3 Methylation. Cell Reports. 2015;10:29–38. doi: 10.1016/j.celrep.2014.12.001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref027] 27.Jones M, Beuron F, Borg A, Nans A, Earl CP, Briggs DC, et al. Cryo-EM structures of the XPF-ERCC1 endonuclease reveal how DNA-junction engagement disrupts an auto-inhibited conformation. Nat Commun. 2020;11(1):1120. Epub 20200228. doi: 10.1038/s41467-020-14856-2 ; PubMed Central PMCID: PMC7048804. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref028] 28.Pufall MA, Graves BJ. Autoinhibitory domains: modular effectors of cellular regulation. Annu Rev Cell Dev Biol. 2002;18:421–62. Epub 20020402. doi: 10.1146/annurev.cellbio.18.031502.133614 . [DOI] [PubMed] [Google Scholar]

[pone.0307590.ref029] 29.Gopaul DN, Duyne GD. Structure and mechanism in site-specific recombination. Curr Opin Struct Biol. 1999;9(1):14–20. doi: 10.1016/s0959-440x(99)80003-7 [DOI] [PubMed] [Google Scholar]

[pone.0307590.ref030] 30.Guo F, Gopaul DN, van Duyne GD. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature. 1997;389(6646):40–6. doi: 10.1038/37925 . [DOI] [PubMed] [Google Scholar]

[pone.0307590.ref031] 31.Gopaul DN, Guo F, Van Duyne GD. Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. Embo J. 1998;17(14):4175–87. doi: 10.1093/emboj/17.14.4175 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307590.ref032] 32.Ghosh K, Lau CK, Gupta K, Van Duyne GD. Preferential synapsis of loxP sites drives ordered strand exchange in Cre-loxP site-specific recombination. Nat Chem Biol. 2005;1(5):275–82. Epub 2006/01/13. nchembio733 [pii] doi: 10.1038/nchembio733 . [DOI] [PubMed] [Google Scholar]

[pone.0307590.ref033] 33.Unnikrishnan A, Amero C, Yadav DK, Stachowski K, Potter D, Foster MP. DNA binding induces a cis-to-trans switch in Cre recombinase to enable intasome assembly. Proc Natl Acad Sci U S A. 2020;117(40):24849–58. Epub 20200923. doi: 10.1073/pnas.2011448117 ; PubMed Central PMCID: PMC7547242. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

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

Shu Hui Huang

Kayla Abrametz

Siobhan L McGrath

Kerri Kobryn

Roles

Abstract

Introduction

Materials and methods

DNAs

Proteins

Telomere resolution assays

Half-site cleavage and hairpin fusion assays

Site-specific recombination assays

Results

Modeling of potential autoinhibitory interfaces within TelA

Fig 1. Structural modeling of potential TelA autoinhibitory interactions.

Mutation of interfaces 1 & 2 stimulates telomere resolution

Fig 2. Telomere resolution assays of interface 1 & 2 mutants.

Combining activating mutations leads to additive activation of TelA

Fig 3. Telomere resolution assays of interface 1 & 2 double mutants.

Testing the modeled autoinhibitory interface interactions with paired charge reversal mutagenesis

Fig 4. Telomere resolution assays of paired charge reversal mutants.

Testing the modeled autoinhibitory interface 1 interactions with deletion analysis and N-terminal domain add back experiments

Fig 5. Telomere resolution by TelA N-terminal domain deletion mutant combined with the interface 1 mutant E337K.

Does hyperactivation of TelA unleash competing or inappropriate activities?

Fig 6. Assessing the ability of hyperactive TelA mutants to promote aberrant reactions.

Fig 7. Assessing the ability of hyperactive TelA mutants to promote reaction reversal.

Testing the most useful hyperactive mutants at standard reaction temperature

Fig 8. Telomere resolution assays of hyperactive mutants at low protein concentration.

Table 1. Summary of mutant phenotypes.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Arthur J Lustig

Roles

Author response to Decision Letter 0

Decision Letter 1

Arthur J Lustig

Roles

Acceptance letter

Arthur J Lustig

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases