Wednesday, November 4, 2009

Telomeres without telomerase in Borrelia spirochetes

ResearchBlogging.orgYou've all heard by now that the 2009 Nobel Prize in Physiology or Medicine will be awarded to Elizabeth Blackburn, Carol Greider, and Jack Szostak. They're the ones who figured out that an enzyme called telomerase combats the shortening that occurs at the ends of the linear chromosomes of eukaryotes (including ours) during each round of DNA replication. Telomerase sticks copies of a short string of nucleotides to the 3' ends of the chromosomal DNA. On the other hand, bacteria do not need telomerase because their chromosomes are circular; they do not have ends that can be shortened.

Spirochetes of the genus Borrelia, which include the agents of Lyme disease and relapsing fever, are an oddity in the bacterial world in that their chromosomes are linear. They also have a large set of linear plasmids. For example, the genome of the Lyme disease agent Borrelia burgdorferi consists of one linear chromosome and 12 linear plasmids, along with 11 circular plasmids. I will refer to the chromosomes and plasmids collectively as replicons. Despite having linear replicons, telomerase is nowhere to be found in Borrelia.

If they lack telomerase, how do Borrelia avoid having the ends of their linear replicons getting pruned during DNA replication? The key lies in the covalently closed hairpin ends of their linear replicons, something that's not found in eukaryotic telomeres. As illustrated in the figure below (figure 1 from Tourand 2003), the hairpin loops allow the replication machinery to copy the nucleotides at the very ends of the telomeres.

The replicated structure shown at the bottom of the figure illustrates that this method of replication creates a new problem. Following DNA replication, the two copies of the replicon end up fused at their ends (L'L and RR'). The fused sequences (telomere junctions) must somehow separate so that one copy of the replicon ends up in each daughter cell as the spirochete lengthens and splits in two.

Fortunately, Borrelia possesses an enzyme designated ResT, a telomere resolvase that cleaves the DNA where the two copies of the replicon are fused and reforms the hairpins at the ends of the new telomeres (see figure below). ResT was discovered by George Chaconas' laboratory in Canada. All of the work with ResT that I describe here was carried out by his group.


Chaconas' group was able to demonstrate the telomere resolvase reaction in vitro by simply mixing purified ResT with DNA containing a telomere junction. The products of the reaction were then analyzed following agarose gel electrophoresis. An example of the assay is shown below. A 4.6 kb piece of DNA containing the telomere junction formed by the left end of the B. burgdorferi linear plasmid lp17 ("L'L") is converted by ResT into the expected 2.6 and 2.0 kb products over a 2 minute time period.

Although the genome sequence of B. burgdorferi was published years ago, the sequence of the telomeres could not be determined back then because of the difficulty in cloning DNA with closed hairpin ends. The sequence of most of the telomeres finally appeared in the literature this year. The telomere sequences are aligned in the table below (figure 6 of Tourand 2009). The end of each telomere is the first nucleotide in each sequence.

The alignment shows that all of the telomeres have a "box 3" sequence. Box 3 is the recognition site for ResT binding. The evidence for this is that ResT binds to box 3 in vitro and changing the nucleotides in box 3 inhibits resolution of the telomere junction by ResT.

The telomeres were grouped based on the box 1 sequence. Type 1 telomeres carry the box 1 sequence TATAAT, and Type 2 telomeres harbor the modified box 1 sequence TATTAT. Type 3 telomeres lack the box 1 motif. When the rates of ResT resolving the different telomere junctions were measured in vitro (last column in alignment above), telomeres that lacked box 1 (Type 3) exhibited the slowest rates, with three telomeres failing to react with ResT. Since these three telomeres are obviously resolved in vivo, their resolution may require additional factors yet to be identified.

References

Tourand, Y., Deneke, J., Moriarty, T.J., & Chaconas, G. (2009). Characterization and in vitro reaction properties of 19 unique hairpin telomeres from the linear plasmids of the Lyme disease spirochete Journal of Biological Chemistry, 284 (11), 7264-7272 DOI: 10.1074/jbc.M808918200

Kobryn, K., & Chaconas, G. (2002). ResT, a telomere resolvase encoded by the Lyme disease spirochete Molecular Cell, 9 (1), 195-201 DOI: 10.1016/S1097-2765(01)00433-6

Tourand, Y., Kobryn, K., & Chaconas, G. (2003). Sequence-specific recognition but position-dependent cleavage of two distinct telomeres by the Borrelia burgdorferi telomere resolvase, ResT Molecular Microbiology, 48 (4), 901-911 DOI: 10.1046/j.1365-2958.2003.03485.x

Tourand, Y., Lee, L., & Chaconas, G. (2007). Telomere resolution by Borrelia burgdorferi ResT through the collaborative efforts of tethered DNA binding domains Molecular Microbiology, 64 (3), 580-590 DOI: 10.1111/j.1365-2958.2007.05691.x