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. 2023 Nov 29;18(11):e0294732.
doi: 10.1371/journal.pone.0294732. eCollection 2023.

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

Mahrokh Balouchi  1 , Shu Hui Huang  1 , Siobhan L McGrath  2 , Kerri Kobryn  1
Affiliations

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

Mahrokh Balouchi et al. PLoS One. .

Abstract

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

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The replication and telomere resolution cycle.
Linear replicons terminated by hp telomeres have DNA replication initiated at an internal origin of replication. This sends out replication forks towards each hp telomere. The replication forks round the hp telomeres producing a circular inverted repeat dimer. In order to segregate the copied DNA into daughter cells a DNA breakage and rejoining reaction referred to as telomere resolution is required to regenerate linear DNA’s terminated by hp telomeres.
Fig 2
Fig 2. Models of telomere resolution promoted by different systems.
TelK: Binding to the rTel leads to dimerization of TelK. TelK induces a large, out of plane, bend in the substrate DNA mediated by the stirrup domain. TelK also induces distortion in the DNA between the scissile phosphates causing buckling of the basepairs in preparation for basepairing to be broken. DNA cleavage allows the energy stored in the DNA bend and distortion to dissolve the dimer. Spontaneous hairpin formation follows once the dimer falls apart. This dimer dissolution seems to be necessary since there is not enough space within the context of an intact TelK dimer for strand refolding into a hairpin conformation [14]. TelA: Binding to the rTel induces dimerization of TelA. It is not known if TelA binding induces a bend in the substrate. Details of steps prior to DNA cleavage are unknown but at some stage of the reaction basepairing must be broken to allow hairpin formation. Post-cleavage stabilization of a strand refolding intermediate is mediated by TelA-DNA contacts and by unexpected non-canonical basepair formation between the refolding strands on the way to hairpin formation. The hairpin products remain engaged with the TelA dimer and are stabilized by numerous TelA-DNA contacts between the scissile phosphates [16]. ResT: Binding to the rTel induces dimerization of ResT and allows engagement of the hairpin-binding module and catalytic domain to cooperate in inducing an underwound conformation of the DNA between the scissile phosphates that licenses ResT to cleave the DNA. An uncharacterized strand refolding process occurs in the context of an intact dimer [17, 18] that leads to hp telomere formation. Hairpin telomere products are released after both hp telomeres are formed [17, 18].
Fig 3
Fig 3. The cold-sensitivity of TelA-promoted telomere resolution is rescued by substrates with mismatches between the scissile phosphates.
A) Schematic of the model rTel used. To aid annealing of the oligonucleotides into an rTel, in preference to hairpins, the rTel is designed with differing lengths of non-telomeric sequence flanking the telomeric sequence (sequence represented with lines flanking the telomeric sequence). This also allows the products of telomere resolution on the two sides of the rTel to be distinguished from each other. The sequence between the scissile phosphates is indicated in red script and the nucleotides are numbered. Nucleotides changed are indicated with red script. B) DNA sequence between the scissile phosphates for the parental rTel and a range of substrate rTels with mismatches (MM) or compensatory mutations (mut) that restore basepairing is shown. C) Comparison of the initial rates of DNA cleavage and total reaction (cleavage + hp formation) of the parental rTel and the mismatch rTels incubated at 30°C (top left graph) compared to reactions incubated at 12°C (top right graph). Also shown is a comparison of the initial rates of cleavage and total reaction of telomere resolution reactions performed with parental rTel and the range of mutant rTels that restore basepairing using 30°C vs. 12°C reaction temperatures (bottom graphs). The initial rates are plotted as the fraction substrate converted/min (1.0 being 100% conversion in one minute). The mean and standard deviation of 3 independent trials is shown.
Fig 4
Fig 4. The cold-sensitivity of TelA-promoted telomere resolution is not rescued by substrates with missing bases between the scissile phosphates.
A) DNA sequence between the scissile phosphates for the parental rTel and a range of substrate rTels with missing bases between the scissile phosphates. The DNA backbone is intact but the base at the positions marked with the red X’s incorporate abasic modifications. B) Comparison of the initial rates of DNA cleavage and total reaction of the parental rTel and the abasic rTels incubated at 30°C (top) compared to reactions incubated at 12°C (bottom). The mean and standard deviation of 3 independent trials is shown.
Fig 5
Fig 5. Characterization of TelA mutants defective for telomere resolution at 30°C using substrates with mismatches between the scissile phosphates.
A) DNA sequence between the scissile phosphates for the parental rTel and a range of substrate rTels with mismatches (MM) or compensatory mutations (mut) that restore basepairing is shown. B) Comparison of the initial rates of DNA cleavage and total reaction of the parental rTel and the mismatch rTels of the indicated TelA mutants conducted at 30°C. C) Comparison of the initial rates of DNA cleavage and total reaction of the parental rTel and the mutant rTels of the indicated TelA mutants conducted at 30°C. The mean and standard deviation of 3 independent trials is shown.
Fig 6
Fig 6. Characterization of TelA mutants defective for telomere resolution at 30°C using substrates with missing bases between the scissile phosphates.
A) DNA sequence between the scissile phosphates for the parental rTel and a range of substrate rTels with missing bases between the scissile phosphates. B) Comparison of the initial rates of DNA cleavage and total reaction of the parental rTel and the abasic rTels using the indicated TelA mutants. The reactions were incubated at 30°C. The mean and standard deviation of 3 independent trials is shown.
Fig 7
Fig 7. Characterization of cold-sensitive TelA mutants using substrates with mismatches between the scissile phosphates.
A) DNA sequence between the scissile phosphates for the parental rTel and a range of substrate rTels with mismatches (MM) or compensatory mutations (mut) that restore basepairing is shown. Nucleotides changed from parental are shown in red script. B) Comparison of the initial rates of DNA cleavage and total reaction of the parental rTel and the mismatch rTels of the indicated cold-sensitive TelA mutants incubated at of 12°C. C) Comparison of the initial rates of DNA cleavage and total reaction of the parental rTel and the mutant rTels of the indicated cold-sensitive TelA mutants incubated at 12°C. The mean and standard deviation of 3 independent trials is shown.
Fig 8
Fig 8. Assessing the cleavage competence of TelA mutants defective for telomere resolution at 30°C via half-site cleavage assays.
A) Schematic outlining the parental and MM1C half-sites used to query TelA cleavage competence. The parental half-site when cleaved produces a transient cleavage product (CP) with a self-complementary 5’-overhang after the distal T1 at the scissile phosphate has diffused away. The self-complementary overhang is refolded into a hairpin conformation and strand resealing occurs. The MM1C half-site has T1 replaced with C1 in the overhang, blocking subsequent hairpin formation, while the diffusion away of T1 on the top strand prevents regeneration of the substrate trapping the cleaved half-site as cleavage products (CP). B) 8% PAGE, 1X TBE, 6M Urea, and 0.1% SDS gel panels of wild type TelA and the 4 TelA mutants defective for telomere resolution with parental rTel incubated at 30°C. S denotes the migration position of the substrate half-site in the gels; hp denotes the hp telomere product and CP denotes cleavage products. The gel labels above the gel indicate the half-site used and time of incubation; M denotes mock reactions, without added TelA, incubated for 120 min.
Fig 9
Fig 9. Assessing the cleavage competence of TelA mutants defective for telomere resolution at 12°C via half-site cleavage assays.
8% PAGE, 1X TBE, 6M Urea, and 0.1% SDS gel panels of wild type TelA and the 4 cold-sensitive TelA mutants defective for telomere resolution with parental rTel incubated at 12°C. Gel labels are as reported in the legend to Fig 8.
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Grants and funding

KK was funded by the Natural Science and Engineering Research Council of Canada (NSERC) grant RGPIN 04382-2017 SHH is funded by the College of Medicine Research award (CoMRAD) grant 426556 SLM was funded the College of Medicine Research award (CoMRAD) grant 424087 MB was funded by a College of Medicine Graduate Research award (CoMGRAD) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.