In Vivo Demonstration of Telomere Resolution

The discovery of a bidirectional, internal origin of replication for the B. burgdorferi chromosome, coupled with the absence of detectable initiation from the hp telomeres, strongly implied that replication would eventually proceed around the hairpin turns to produce inverted repeat replicated telomere (rTel) junctions. A significant advance in our understanding of the mode of replication of the linear genetic elements came from the subsequent demonstration in vivo that such rTel junctions are processed by a DNA breakage and reunion reaction, referred to as telomere resolution, to produce two hp telomeres (Fig. 1; Chaconas et al. 2001). In this study it was demonstrated that integration of a synthetic rTel into lp17, near the left telom-ere, creates a substrate for a cellular telomere resolvase activity that acts at the rTel to produce a deletion derivative of lp17 containing a bona fide hp telomere derived from the synthetic junction. The part of lp17 deleted from the transient lp17 integrant species is lost from the cell, presumably due to the lack of replication functions on the resulting fragment, providing further evidence that hp telomeres cannot serve as origins of replication. More evidence that the synthetic rTels are resolved into hp telomeres comes from an experiment which showed that cloning of a synthetic rTel into a circular shuttle vector (pBSV2) replicating in B. burgdorferi is sufficient to convert the shuttle vector into a linear replicon terminated by hp telomeres (Chaconas et al. 2001). Telomere resolution in vivo is sequence specific; a mock rTel junction with the same inverted repeat symmetry and sequence composition of a real substrate, but that is of non-telomeric sequence, is not a substrate for the telomere resolvase. Another noteworthy result is that integration of an rTel into lp17 is accompanied by a 7- to 15-fold stimulation of the recovery of transformants, implying that either rTel junctions or hp telomeres are intrinsically recombinogenic. This effect is dependent upon the rTel being resolvable, suggesting that the stimulation of recombination is a consequence of the action of the telomere resolving machinery (Chaconas et al. 2001).

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