Abstract
The genome of the Lyme disease pathogen Borrelia burgdorferi contains about a dozen linear DNA molecules that carry covalently closed hairpin telomeres as a specialized mechanism for dealing with the end-replication problem. The hairpin telomeres are generated from replicative intermediates through a two-step transesterification promoted by the telomere resolvase ResT. Although the genome of B. burgdorferi has been sequenced, the sequence of most telomeres has remained unknown because of difficulties in recovering and completely sequencing the covalently closed hairpin ends. In this study we report a new approach for the direct sequencing Borrelia telomeres and report the sequence, characterization, and in vitro reaction properties of 19 unique telomeres. Surprisingly, a variation of greater than 160-fold in the initial reaction rates of in vitro ResT-mediated telomere resolution was observed between the most active and least active telomeres. Moreover, three of the hairpin telomeres were completely inactive in vitro, but their in vivo functionality was demonstrated. Our results provide important new information on the structure and function of the B. burgdorferi telomeres and suggest the possibility that factors besides the telomere resolvase ResT may influence the reaction in vivo and rescue those telomeres that are not functional in vitro with ResT alone.
Highlights
Research Grant MOP-53086, the Canada Research Chairs Program, and the Alberta Heritage Fund for Medical Research
Linear DNA molecules are rarely found in bacteria, and those in B. burgdorferi are terminated by covalently closed hairpin ends [18, 19]
The ResT protein has been the subject of a number of investigations (24, 26 –33), and a crystal structure has been reported for a phage telomere resolvase [34], little information has been available for the telomeres of B. burgdorferi, which is a powerful model organism for studying hairpin telomere biology
Summary
Research Grant MOP-53086, the Canada Research Chairs Program, and the Alberta Heritage Fund for Medical Research. Copying of the DNA on each side of the linear molecule results in the formation of a dimer junction or replicated telomere (LЈL and RRЈ) in the replicative intermediate These regions are subsequently processed in a DNA breakage and reunion reaction referred to as telomere resolution [23], promoted by the telomere resolvase ResT [24]. This essential enzyme [6] performs a two-step transesterification to generate covalently closed hairpin ends from the replicated telomeres using a reaction mechanism similar to that of tyrosine recombinases and type IB topoisomerases (24 –26). The box 3–5 region appears to be invariant in position in all telomeres studied to date with the qualification that the position of the
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