Fig. 2. Construction of linear plasmid integrants and predicted structure of telomere resolution products. (A) An E.coli plasmid (pKK81) carrying bp 1424–3315 from the left end of lp17 was used in a B.burgdorferi transformation to generate a plasmid integrant in lp17 via homologous recombin ation. (B) A linear plasmid integrant was constructed as in (A), but with an E.coli plasmid (pGCL10-2) that also carried the 140 bp replicated left-end telomere of lp17 corresponding to the L′–L junction (Figure 1). DNA breakage and reunion in the replicated telomere are expected to give rise to the two linear molecules shown; only the larger plasmid would contain the gene for kanamycin resistance (kan). Although the diagram has been drawn with homologous recombination preceding telomere resolution, a specific temporal order of events is not implied; telomere resolution of the transforming DNA followed by homologous recombination would yield the same set of final products. Blue denotes the kan gene, yellow represents the E.coli plasmid vector and green the lp17 sequences used for the recombination target. White indicates other lp17 sequences and red denotes telomeric regions. Probes 1 and 2 indicate the regions used as hybridization probes in Figure 3. The schematic is not drawn to scale.

Fig. 4. Telomere structure of lp17 constructs in B.burgdorferi transformants. (A) Predicted PCR products using left-end primer OGCB5 and kan primer pOK.2 are shown above each telomere. The hatched region indicates the kan gene. (B) Ethidium bromide-stained 1.4% agarose gel containing PCR reactions using the indicated DNAs. The sizes (in bp) of molecular weight markers in lane M are indicated.

Fig. 6. Conversion of a circular plasmid to a linear form. (A) Schematic showing the shuttle vector without (pBSV2) or with (pGCL40-5) the 140 bp replicated telomere (L′–L) inserted into the polylinker (solid line). The circular plasmid pGCL40-5 is expected to be converted to a linear form if telomere resolution occurs. (B) Southern blot of a 0.7% agarose gel containing total B.burgdorferi plasmid DNA from strain B31-A transformed with the circular shuttle vector (pBSV2) or the shuttle vector carrying the replicated telomere (pGCL40-5). DNA samples were run before and after digestion with NcoI. Five times more DNA was used for the pGCL40-5 transformant because of the 5-fold reduction in copy number. The blot was hybridized with a probe made from the backbone (pOZK) of the shuttle vector (Stewart et al., 2001). The migration positions of the supercoiled, linear and relaxed forms of pBSV2 are denoted by S, L and R, respectively. The migration positions of 1.68 and 4.73 kb markers (not shown) are noted on the right side of the figure.

Fig. 1. Two models for replication of linear DNA with hairpin ends. The arrows labeled L and R indicate the left and right inverted repeats, respectively, at the telomeres of linear Borrelia DNA. In pathway A, the line bisecting the telomere junctions in the replicated dimer carrying head-to-head (L′–L) and tail-to-tail (R–R′) junctions is an axis of 180° rotational symmetry. In pathway B, the closed circles denote 5′ phosphates at nick sites or at the DNA ends. A more complete set of models can be found in Casjens (1999).

Fig. 3. Analysis of plasmid DNA from B.burgdorferi transformants. Left panel: an ethidium bromide-stained field inversion gel of plasmid DNA from untransformed B31-A (lane 1) or from B31-A transformed with pKK81 (lane 2; minus the lp17 telomere), pGCL10-2 (lane 3; plus the 140 bp replicated telomere) and pGCL13–1 (lane 4; with a 140 bp replicated mock telomere). The migration positions of 48.5 and 23.1 kb markers and wild-type lp17 are shown. Center panel: a Southern blot of the gel in the left panel, hybridized with Probe 1, corresponding to bp 414–1231 from the left end of lp17 (Figure 2). Right panel: a Southern blot of the gel in the left panel, hybridized with Probe 2 from the kan gene. The migration positions of intact lp17 integrants (I), resolved integrants (R) and wild-type lp17 (Wt) are shown on the right side of the figure.

Fig. 5. Test for hairpin telomere at the left end in the resolved substrate. (A) The schematic shows the telomeric structure of wild-type lp17 and the position of an EcoNI site located 2639 bp from the left end. The right panel shows a Southern blot of a 1% agarose gel containing EcoNI-digested B.burgdorferi plasmid DNA hybridized with Probe 1. Duplicate samples were loaded without (–) or with (+) heat treatment at 95°C for 6 min, followed by incubation at 0°C, as indicated. (B) The schematic shows the position of an XhoI site 2538 bp from the left end of the resolved telomere substrate. The blot on the right contains XhoI-digested plasmid DNA from a B.burgdorferi transformant carrying the resolved telomere substrate. The blot was hybridized with Probe 2 (kan). (C) The schematic shows the resolved telomere substrate cut with both XhoI and BamHI to remove the hairpin end on a 70 bp fragment. The blot on the right was also hybridized with Probe 2.