1. Fusion of Hairpin Telomeres by the B
Fusion of Hairpin Telomeres by the B. burgdorferi Telomere Resolvase ResT 
Kerri Kobryn, George Chaconas 
Molecular Cell 
Volume 17, Issue 6, Pages (March 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
The Replication Pathway for Linear DNA Replicons with Covalently Closed Hairpin Ends
The L and R arrows indicate the DNA hairpin telomeres at the left and right ends, respectively. The lines bisecting the head-to-head (L′–L) and the tail-to-tail (R–R′) replicated telomere junctions are axes of 180° rotational symmetry. It is not yet known whether replication is completed before telomere resolution (as shown) or whether processing at either end can occur before replication is completed at the other. This figure has been adapted from (Kobryn and Chaconas, 2002). A similar replication strategy has been demonstrated for the E. coli phage N15 (Ravin, 2003; Ravin et al., 2003).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Hairpin Inhibition of Telomere Resolution by ResT
(A) Autoradiograph documenting the inhibition of telomere resolution by various concentrations of hairpin telomere (hp) or replicated telomere. 3′-labeled, BglII-digested pYT11 substrate was present at 0.8 nM and incubated with 37 nM ResT at 30°C for 15 min in the presence of the indicated amounts of unlabeled hp or rtel competitor DNAs. The hp competitor was obtained by prior resolution of unlabeled BgllI-digested pYT11 substrate plasmid and the rtel competitor by addition of unlabeled BgllI-digested pYT11. A nonsubstrate plasmid (pSD7) was used as the nonspecific DNA competitor. Products were analyzed by agarose gel electrophoresis (1% agarose, 1× TAE) followed by autoradiography after the gel was dried. A schematic of the substrate plasmid (rtel) and the larger of the two hairpin (hp) products are shown at the left of (A).
(B) Graphical representation of the data from (A). The competitor-free reaction proceeded to an absolute value of 74% resolution, which was used as the 100% reference point to which the remaining reactions were normalized.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Cleavage of Hairpin Product Telomeres by ResT
(A) Schematic of an OPS-modified hairpin telomere used to trap cleaved ResT-DNA intermediates as a covalent protein-DNA complex (CPD). The OPS hp is a 25 bp cleavable but nonligatable product mimic in which the 5′ bridging oxygen at the cleavage site has been substituted by sulfur. For simplicity the nonbridging oxygens at the cleavage site were omitted from the schematic. Abbreviations: asterisk, 5′ 32P label; Y, tyrosine 335 nucleophile; hp, hairpin DNA.
(B) Autoradiograph of SDS-PAGE (8%) analysis of ResT-mediated hairpin cleavage. Reactions were incubated at 30°C for 30 min with 8 nM hairpin telomere and 37 nM ResT. The reactions were terminated by addition of loading dye supplemented with SDS to a final concentration of 0.3%. Protease treatment was performed in the loading dye with 500 units of pronase. YF = the ResT Y335F mutant in which the active site tyrosine nucleophile has been changed to phenylalanine. M, marker of a 50 bp replicated telomere (rtel).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Reversal of the Telomere Resolution Reaction by ResT
(A) Experimental design to detect and characterize the reverse reaction. Abbreviations: asterisk, 5′ 32P label; hp, hairpin DNA; rtel, replicated telomere.
(B) Autoradiographs of native and denaturing PAGE analysis of reverse telomere resolution. Reactions were incubated at 30°C for 30 min with 8 nM labeled 35 bp hairpin, 8 nM unlabeled 70 bp hairpin (where indicated) and 74 nM ResT. Reactions loaded onto the native gel were stopped and pronase treated as reported for Figure 3. The native gel was 8% PAGE, 1× TAE. Reactions loaded onto the denaturing 8% TBE/urea gel were terminated by addition of formamide loading dye and by heating to 95°C for 5 min prior to loading. Abbreviations: hp, DNA hairpin; mock, a 70 bp hairpin telomere of nontelomeric sequence; YF, ResT Y335F.
(C) Schematic of the reverse telomere resolution reaction. The reverse reaction is shown as a two-step transesterification where two hairpin telomeres are first transesterified to tyrosine 335 of ResT. Two such hairpins can then be covalently linked to form a replicated telomere by the second transesterification, where the free 5′OH end of each cleaved hairpin performs a nuclophyllic attack on the phosphotyrosine linkage of the other cleaved hairpin.
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6. Figure 5
Reversal of the Telomere Resolution Reaction on a Plasmid Substrate
(A) Schematic of telomere resolution on a plasmid substrate and the products from subsequent reaction reversal. The position of the replicated telomere junction is indicated by the black box bisected by a line. Abbreviations: S, 4.6 kb linear substrate; P1, 2.6 kb product; P2, 2.0 kb product; P1 + P1, 5.2 kb reversal product; P1 + P2, 4.6 kb reversal product; P2 + P2, 4.0 kb reversal product.
(B) Agarose gel analysis of reaction reversal on a plasmid substrate. Pst I-digested pYT11 (3.2 nM) was incubated at 30°C with 148 nM ResT and aliquots removed at the indicated time points. Reversal products from a separate large-scale overnight reaction were gel purified and incubated at 30°C ± 148 nM ResT for 40 min.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Low-Temperature Incubation Favors the Reverse Reaction (Telomere Fusion)
Autoradiograph of native PAGE analysis of the forward and reverse reactions run at different temperatures. 40 nM, 5′-labeled 50 bp replicated telomere (rtel, forward reaction) or 25 bp hairpin telomere (hp, reverse reaction) were incubated at the indicated temperatures for 16 hr with 148 nM ResT. The reactions were pronase treated prior to being loaded onto an 8% 1× TAE PAGE gel. The direction of the reaction that was assayed is based upon the input substrate and is noted below the gel. Abbreviations: hp, hairpin telomere; rtel, replicated telomere; YF, Y335F active site mutant.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Telomere Exchange by ResT-Mediated Telomere Fusion
The proposed mechanism for telomere exchange between linear plasmids and the right end of the B. burgdorferi chromosome requires a telomere fusion event that is stabilized by deletion formation to inactivate or remove the newly fused telomere and prevent its resolution. The telomere fusion event is promoted by reversal of the ResT reaction such that two hairpin telomeres from different molecules are fused to generate a single DNA molecule carrying a replicated telomere. Formation of a deletion, which removes the telomere resolution site, might be specifically targeted to the fused telomere by incomplete joining in the reverse reaction to leave a ResT molecule covalently linked at a nick in the telomere; such covalent protein-DNA complexes are known to be foci for the formation of deletions and other chromosomal aberrations (Froelich-Ammon and Osheroff, 1995). Alternatively, the deletion could be derived from palindrome instability induced by passage of a replication fork through the inverted repeat of the fused telomere (Leach et al., 1997; Pinder et al., 1998).
The example shown in Fusion 1 links a linear plasmid (lpX) to the right end of the B. burgdorferi R-IP3 chromosome to generate the structure of the right end telomere found in the B31 chromosome. The identitiy of lpX is not clearly discernible, and the right end of the B31 chromosome shares homology with several linear plasmids (Casjens et al., 1997; Casjens et al., 2000). Fusion 2 shows a telomere exchange that converts the right end of B31 to the right end observed for the Sh-2-82 chromosome through fusion with lp21 (see Casjens et al., [1997]; Huang et al., [2004b]). Successive rounds of telomere fusion with deletion formation can also explain the many examples of telomere exchange observed in the B. burgdorferi linear plasmids (Casjens et al., 2000).
Molecular Cell  , DOI: ( /j.molcel )
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