Fig 1.
Telomere resolution and Cre-like recombination promoted by TelA.
A) Telomere resolution involves dimerization of TelA on a replicated telomere (rTel) junction followed by a pair of transesterifications that result in DNA cleavage and strand rejoining (after refolding the cleaved strands) to form the hairpin (hp) telomere products. The reaction is promoted by the active site nucleophilic tyrosine (shown with a Y) using a mechanism similar to that used by type IB topoisomerases and tyrosine recombinases. B) Cre-like site-specific recombination involves dimerization of TelA on a pair of rTels followed by their synapsis. DNA cleavage of one scissile phosphate in each rTel followed by strand exchange leads to the formation of a Holliday junction (HJ) that is normally transiently present intermediate of recombination promoted by tyrosine recombinases. The HJ is isomerized and undergoes a further round of DNA cleavage and strand transfer events using the second pair of scissile phosphates to produce a full, directly repeated, recombinant dimer. TelA tends to accumulate the HJ as its predominant product. C) Shown are graphics of the rTel-containing plasmid used in recombination reactions and the predominant HJ intermediate and recombinant dimer produced by reaction. For simplicity only the HJ and recombinant dimer versions of Cre-like recombination from an anti-parallel synapse are shown.
Fig 2.
Determination of the minimal requirements for TelA hyperactivation and conversion into a site-specific recombinase.
A) The sequences of the replicated telomere junction (rTel) and the telomeric half-site are shown. The half-sites are made up of one copy of the symmetry element that makes up the inverted repeat found in the rTel. Mock versions systematically changed A’s to T’s, G’s to C’s (and vice versa) to maintain the sequence composition and secondary structure while changing the sequence of the substrates. These sequences were directionally cloned into pUC19 as detailed in the Materials and Methods section. B) 0.8% agarose 1X TAE gel panels of incubations with 50 nM of the noted TelA mutants vs. wild type TelA using 2 µg/mL of pUC19 variants with telomeric half-site and rTel inserts. Also shown are control panels with mock half-site and mock rTel versions of the plasmids. All incubations were at 30oC for 30 min in a buffer containing 2 mM MgCl2. S denotes substrate; P1 & P2 denote the products of telomere resolution; topo denotes the ladder of topoisomers; rec? denotes plasmid multimers likely composed of HJ’s and full recombinants. For labeling the mutants shown above the gel-loading key the triple mutant shortens the D202R/E337K portion of the triple mutant to DREK. C) Summary graph of the initial rates of telomere resolution vs. recombination for the recombination proficient mutants tested with 50 nM TelA. D) Summary graph of the initial rates of telomere resolution vs. recombination of the mutants lacking the D398A mutation needed to activate recombination; the mutants were tested using 50 nM TelA. NA indicates that no activity was detectable at the longest timepoint tested (2 h). Shown are the mean and standard deviation of three independent trials of each experiment.
Fig 3.
Analyzing the hyperactive recombinases for their responsiveness to divalent metal-ion concentration.
A) Schematics of the structure of the plasmid substrates used for the recombination, topoisomerase and telomere resolution assays, respectively. The position of the single-hit restriction site for SspI relative to the position of the cloned rTel and half-site inserts is shown. The inserts divide the plasmid substrates into 2.1 kb and 0.6 kb domains relative to the position of the SspI site. The sequence of the rTel and telomeric half-site inserts present in the plasmids is detailed in Fig 2A. B) 0.8% agarose 1X TAE gel panels of divalent metal ion titrations of the D202R/D398A and D202R/E337K/D398A mutants reacted with negatively supercoiled rTel plasmid substrate (pEKK494) incubated at 30oC for 30 min in a buffer containing the indicated concentrations of MgCl2. The 0, or no Mg2+ condition, contains 1 mM EDTA instead. C) 0.8% agarose 1X TAE gel panel of divalent metal ion titrations of the D202R/D398A and D202R/E337K/D398A mutants reacted with negatively supercoiled telomeric half-site plasmid substrate (pEKK588) incubated at 30oC for 30 min in a buffer containing the indicated concentrations of MgCl2. The 0, or no Mg2+ condition contains 1 mM EDTA instead. S denotes substrate; rec denotes multimers of recombined plasmids. D) 0.8% agarose 1X TAE gel panels of divalent metal ion titrations of the D202R/D398A and D202R/E337K/D398A mutants reacted in telomere resolution reactions with SspI-linearized wildtype plasmid substrate (pEKK494) at 30oC for 10 min and 30 min timepoints in a buffer containing the indicated concentrations of MgCl2. S denotes substrate; P1 & P2 denote the migration position of the expected products of telomere resolution.
Fig 4.
Analysis of the HJ produced by the recombinase mutants with plasmid reactions: determining if asymmetrizing the rTel sequence allows for reaction directionality control.
A) Representation of the expected structure of the HJs resulting from alternate orientations of synapsis and strand exchange. Also shown are the expected results from incubation with a HJ resolvase (T7 endo I) in the cases where two strands or four strands of the HJ have been cleaved. A version of the graphic originally appeared in our previous paper, held under a creative commons license (ref [12]). B) Shown is the sequence between the scissile phosphates for the parental rTel sequence with complete inverted repeat symmetry vs. two mutant rTels that disrupt the inverted repeat symmetry of the parent via mutation(s) introduced between the scissile phosphates (mutant rTel 1; pEKK495) and (mutant rTel 2; pEKK592). C) 0.8% agarose 1X TAE gel panel of the reaction of the D202R/D398A and D202R/E337K/D398A mutants with the parental rTel (pEKK494) employing 30oC, 30 min and 60 min incubations, respectively, in a buffer with 2 mM MgCl2 followed by digestion of the reactions with SspI and by treatment at 37oC, 10 min with T7 endonuclease I. Gels labels are as noted in D). D) 0.8% agarose 1X TAE gel panel of the reaction of the D202R/D398A and D202R/E337K/D398A mutants with mutant rTel 1(pEKK495) and mutant rTel 2 (pEKK592) employing 30oC, 2 h incubations in a buffer with 2 mM MgCl2 followed by digestion of the reactions with SspI and by treatment at 37oC, 6 min with T7 endonuclease I. M denotes a mock incubation of supercoiled substrate plasmid without TelA addition or subsequent treatment; HJ denotes Holliday junctions; the numbers to the right of the gel indicate the size of the products noted in kb. The ethidium bromide stained gel is shown as an inverted image.
Fig 5.
HJ formation in recombination reactions between plasmid substrates and small synthetic rTels: determining if asymmetrizing the rTel sequence allows for reaction directionality control.
A) Schematic of the reaction between a plasmid carrying an rTel junction and a 5’-fluorescein endlabeled rTel assembled with oligonucleotides. The asterisks (*) indicate the position of the 5’-fluorescein endlabels. 1) The two possible orientations of the resulting HJs is shown, assuming a non-directional recombination reaction using a wild type rTel junction with complete dyad symmetry. 2) shows the product expected from the HJs when they are disrupted by heating the resultant HJs to 75oC prior to gel loading. This mimics branch migration to a linear recombinant product. A version of the graphic originally appeared in our previous paper, held under a creative commons license (ref [13]). B) 0.8% agarose 1X TAE gel panels of reactions of the TelA (D202RD398A) mutant with pEKK494 (parental rTel) and pEKK592 (mutant rTel 2) with 5’-fluorescein endlabeled small synthetic rTels of the same sequence (with heterologous flanks). The reaction conditions were optimized separately for the two substrate combinations as noted in the Materials and Methods section. scHJ; denotes a supercoiled HJ formed between the oligo rTel and plasmid rTel (also the migration position of supercoiled substrate plasmid), linrec; denotes the linear product, with a fluorescein label, of heat disruption of the scHJ, scHJdim; denotes the migration position of the product of strand exchange between plasmids to make a dimer, 75C; denotes the samples that were heat treated prior to gel-loading. Beneath the gel panels is shown the identity of the recombining rTels. The sequence of the rTels is shown in Fig 4B. C) 0.8% agarose 1X alkaline gel panel of reactions of the TelA (D202RD398A) mutant with pEKK494 (parental rTel) with a 5’-fluorescein endlabeled small synthetic rTel of the same sequence (with heterologous flanks). The strand lengths (in kilonucleotides) are noted on the sides of the gel. The 1 kb ladder was cropped and ethidium bromide stained for imaging while the remainder of the gel was not stained but instead visualized using the fluorescein channel. A composite image of the ladder and experiment is presented.
Fig 6.
Analysis of the TelA mutants activated for telomere resolution.
A) Summary graph of the initial rates of telomere resolution for wild type TelA (WT) and the hyperactive TelA mutants plotted against TelA concentration. The buffer conditions utilized include 2 mM MgCl2. Shown are the mean and standard deviation of three independent trials of each experiment. B) Summary graph of the initial rates of telomere resolution with wild type TelA and the hyperactive mutants plotted for reactions containing 12.5 nM and 6.25 nM TelA comparing reaction rates obtained in buffers containing 1mM EDTA vs. 2 mM MgCl2. Shown are the mean and standard deviation of three independent trials of each experiment.
Fig 7.
Testing the ability of the TelA mutants to promote reaction reversal.
A) Half-site cleavage assay of the TelA mutants. Left: shown is a graphic depicting the cleavage assay. When a telomeric half-site is employed TelA cleavage results in a covalent linkage of TelA to the DNA that can be visualized as a bandshift in SDS-containing polyacrylamide (SDS-PAGE) gels. The asterisk indicates a 5’-FITC based fluorophore for visualization of the DNA in the gels. Right: 8% PAGE 1X TAE/0.1% SDS gels of reactions containing 150 nM TelA incubated at 5oC for 17 h. The numbers above the substrate bands in each lane indicate the percentage of cleavage product obtained in the reaction. hs denotes the half-site; CP denotes the cleavage product. B) hairpin (hp) telomere fusion assay. Left: shown is a graphic of the hp telomere fusion assay. Fusion of a pair of hp’s leads to formation of a replicated telomere junction (rTel). This is a reversal of the telomere resolution reaction. Right: 8% PAGE 1X TAE/0.1% SDS gels of reactions containing 150 nM TelA incubated at 5oC for 17 h. The numbers just above the substrate bands in each lane indicate the percentage of reaction reversal obtained in the reaction. hp denotes the hairpin telomere substrate; rTel denotes the replicated telomere product of fusion of two hp telomeres. A version of the graphics originally appeared in our previous paper, held under a creative commons license (ref [13]).