Telomeres are comprised of short G-rich DNA repeats bound by a set of specific proteins that cap the ends of linear chromosomes in eukaryotes. They serve a multitude of purposes including: (a) distinguishing chromosome ends from DNA double-strand breaks, (b) regulating access of telomerase and other enzymes to chromosome ends, (c) protecting the termini from uncontrolled exonucleolytic degradation, (d) participating in nuclear organization by anchoring telomeres in the nuclear periphery, and (e) regulating expression of genes located near chromosome ends.
As many of these processes are conserved among eukaryotes, several projects in our laboratory rely on the fission yeast Schizosaccharomyces pombe as a model organism. We focus on the identification and characterization of factors involved in telomere length regulation, on the specific roles of telomeres during meiosis, and on the mechanism by which telomeres are distinguished from DNA double-strand breaks.
Mechanism of Pot1-mediated chromosome end protection.
Deletion of the pot1+ (Protection of Telomeres) gene causes rapid loss of telomeric DNA leading to chromosome instability and cell death. We identified Pot1 initially in fission yeast (Baumann and Cech, 2001), but subsequently found homologs in many species including other yeasts, microsporidia, plants, and vertebrates (Baumann et al., 2002). Consistent with a direct role of Pot1 in protecting chromosome ends, biochemical studies demonstrated that the protein binds the G-rich strand of telomeric DNA with exceptionally high specificity (Baumann and Cech, 2001; Lei et al., 2002). The crystal structure of a 22 kDa N-terminal Pot1 fragment (Pot1N) in complex with DNA explained this high specificity by a novel form of DNA self-recognition involving base-stacking and unusual G-T base pairs. To verify the validity of this structure in vivo, we generated a series of Pot1 mutants containing single and double amino acid substitutions and expressed those mutant proteins in fission yeast. This work was published in collaboration with Tom Cech’s group who solved the crystal structure of Pot1N (Lei et al., 2003).
Purification and subsequent characterization of full-length Pot1 protein revealed the presence of a second DNA binding site that recognizes DNA with reduced sequence specificity compared with the domain present in Pot1N. The two DNA binding motifs cooperate to mediate binding to telomeric repeats in a specific register. Consistent with a role in chromosome end capping, we showed that Pot1 prevents access of telomerase to the chromosome end and protects specific sequence permutations at the 3’ terminus against exonucleolytic degradation (Trujillo et al., 2005).
Balancing chromosome end protection and telomere elongation.
Using a series of Pot1 truncation mutants, we demonstrated that a large portion of Pot1, including the N-terminal DNA-binding domain and amino acids close to the C-terminus are essential for its protective function in vivo. We found that C-terminal Pot1 fragments exert a dominant negative effect by displacing endogenous Pot1 from telomeres. Reducing telomere-bound Pot1 in this manner resulted in dramatic lengthening of telomeres, hence uncovering a role of Pot1 in telomere length regulation. A further reduction of telomere-bound Pot1 caused rapid loss of telomeric DNA followed by chromosome end fusions. Our results show that cells must carefully regulate the amount of telomere-bound Pot1 to allow regulated access by telomerase, but prevent catastrophic loss of telomeres (Bunch et al., 2005). More recent work suggests that the level of Pot1 protein is indeed tightly regulated and that excess Pot1 is targeted for degradation. We are now investigating the mechanism that controls Pot1 stability as well as the pathway by which Pot1 is degraded.
Identification of Pot1 interacting proteins.
As our biochemical studies had mapped the DNA binding site near the N-terminus of Pot1, we anticipated that this domain would be required and sufficient for telomere localization in vivo. Surprisingly, we found that C-terminal, but not N-terminal Pot1 fragments localize efficiently to telomeres. This result prompted us to search for proteins that interact with Pot1 and may mediate its recruitment to telomeres. We identified several candidates by two hybrid screens using full length Pot1 or C-terminal Pot1 fragments as bait. In parallel, we employed dual affinity purification to isolate Pot1-containing complexes from cell extracts. We are now in the process of validating the interactions biochemically and assessing roles for the identified proteins in telomere maintenance by gene deletion and overexpression analysis.
Activation and recruitment of telomerase.
The machinery which replicates the bulk of chromosomal DNA is intrinsically incapable of fully replicating the chromosome termini. Due to this ‘end-replication problem’ terminal sequences are lost with every round of replication, eventually leading to chromosomal instability and cellular senescence. In the germ line and in unicellular eukaryotic organisms, telomeric DNA is replenished by the enzyme telomerase. However, several studies have shown that expression of the catalytic subunit of telomerase alone is not sufficient for maintaining stable telomeres. We are interested in how telomerase is recruited to chromosome ends and what mechanism maintains telomere length homeostasis.
Which proteins mediate meiosis-specific functions of telomeres?
Telomere clustering and telomere-led chromosome movements are essential for the orderly progression through meiosis, yet their mechanistic basis is only poorly understood. Similarly, we know little about the process by which telomere length is reset prior to gametogenesis. We are taking a biochemical approach to identifying factors that specifically associate with telomeres during the different stages of meiosis. Towards this aim we have generated strains that can be induced to enter meiosis synchronously and contain dual affinity tags on known telomere components. Currently, we are focusing on the identification of proteins that mediate the association between telomeres and the spindle pole body during the horse-tail stage of meiosis.
What factors mediate chromosome end fusions following loss of telomeric DNA?
Telomere shortening eventually results in chromosome ends being perceived as DNA double-strand breaks and being engaged by DNA repair enzymes. Under most conditions, telomere attrition is a slow process and only a small fraction of telomeres reach a critically short length at any one time. Using conditional alleles of Pot1, we are now able to induce synchronous telomere loss, providing a system to analyze the changes in proteins that associate with chromosome ends. We have shown genetically that chromosome end fusions occur in the absence of key factors involved in homologous recombination and non-homologous end-joining. To elucidate the mechanism of end fusions we are combining candidate-driven approaches with genetic screens and protein biochemistry.
References
Baumann, P., and Cech, T. R. (2001). Pot1, the putative telomere end-binding protein in fission yeast and humans. Science 292, 1171-1175.
Baumann, P., Podell, E. R., and Cech, T. R. (2002). Human Pot1 (protection of telomeres) protein: cytolocalization, gene structure, and alternative splicing. Mol Cell Biol 22, 8079-8087.
Bunch, J. T., Bae, N. S., Leonardi, J., and Baumann, P. (2005). Distinct requirements for Pot1 in limiting telomere length and maintaining chromosome stability. Mol Cell Biol 25, 5567-5578.
Lei, M., Baumann, P., and Cech, T. R. (2002). Cooperative Binding of Single-Stranded Telomeric DNA by the Pot1 Protein of Schizosaccharomyces pombe. Biochemistry 41, 14560-14568.
Lei, M., Podell, E. R., Baumann, P., and Cech, T. R. (2003). DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA. Nature 426, 198-203.
Trujillo, K. M., Bunch, J. T., and Baumann, P. (2005). Extended DNA binding site in Pot1 broadens sequence specificity to allow recognition of heterogeneous fission yeast telomeres. J Biol Chem 280, 9119-9128.
Baumann, P. (2006) Are Mouse Telomeres Going to Pot? Cell 126, 33-36.
Xhemalce, B., Riising, E.M., Baumann, P., Dejean, A., Arcangioli, B. and Seeler, J.-S. (2007) Role of SUMO in the dynamics of telomere maintenance in fission yeast. PNAS 104, 893-898.