Regulation of ResT Activity In Vivo

As expected from its central role in the replication of the linear replicons in B. burgdorferi, ResT is an essential cellular activity. Efforts to knock out ResT always result in merodiploidy (Byram et al. 2004) unless a homologue from B. hermsii is first provided to the cell (Tourand et al. 2006). Despite the successful replacement of B. burgdorferi ResT with B. hermsii ResT and the normal growth and genome maintenance in such strains, B. hermsii ResT was found to be unable to process the type 2 rTels in vitro. Closer examination of some of the inactive substrates assayed earlier in vitro for B. burgdorferi ResT also revealed that they are functional in the context of the spirochete (Tourand et al. 2006). This suggests the action of either a redundant function (notwithstanding the essential nature of ResT) or, more likely, the participation of one or more accessory factors in vivo.

Recent results suggest a completely unexpected candidate: positive DNA supercoiling (Bankhead et al. 2006). In vitro results reveal a strong stimulation of telomere resolution by B. burgdorferi ResT and a complete rescue of the resolution defect of B. hermsii ResT on a type 2 rTel. Negative super-coiling has an opposing effect; a progressive inhibition of resolution occurs with increasing levels of negative supercoiling. Biochemical studies indicate that the stimulation from the positive DNA supercoiling is conferred by promoting a very early reaction step: formation of ResT-ResT interactions in a cross-axis complex inferred to exist from the studies discussed in Sect. 5.6 (see Fig. 4 for a schematic summary cartoon of the telomere resolution reaction). The results are most consistent with this interaction being promoted by the twist component of the positive supercoiling (Bankhead et al. 2006). Negative supercoiling plays an important role in many aspects of DNA metabolism in bacteria, including replication, transcription, packaging, recombination and transposition (Kanaar and Cozzarelli 1992). Until now positive DNA su-percoiling has only been viewed in eubacteria as a by-product of processes such as transcription and replication that must be removed by cellular topoi-somerases. These results raise the intriguing possibility that the energy of positive DNA supercoiling per se may be harnessed to activate/control telom-ere resolution, linking telomere resolution to some process that produces

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positive twist or supercoiling in the spirochete (e.g. replication or transcription converging on, as yet, unresolved replicated telomere junctions). Future studies will be required to test this hypothesis, since it is also possible that the positive DNA supercoiling in vitro is merely substituting for an unidentified accessory factor. Figure 4 presents a summary model of the telomere resolution reaction catalysed by ResT.

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