Telomerase, Genome Instability and Chromosome Biology
     Our main research goal is to understand how defects in telomere maintenance contribute to cancerogenesis, aging, and various diseases. A better understanding of the dynamic interactions that occur at the telomere will ultimately enable us to identify compounds that modulate telomere length, either to limit the life span of tumor cells or to boost the proliferative potential of desired cell populations.

Towards this aim, we employ a variety of techniques in biochemistry, molecular genetics, and cell biology to dissect and elucidate the functions of the protein-nucleic acid complexes at human telomeres. As chromosome end protection and telomere maintenance are common challenges for all organisms with linear chromosomes, several projects in our laboratory rely on the fission yeast Schizosaccharomyces pombe as a model organism. Here we focus on telomerase biogenesis, the regulation of telomere length and the mechanism by which chromosome ends fuse when telomeres fail.

A few years ago we became interested in the fact that over 70 species of vertebrates seem to be thriving without males. While it was demonstrated more than 30 years ago that such clonal lineages have originated by cross-hybridization of sexual species, little is known about the molecular mechanism of parthenogenetic reproduction. What started with simple curiosity in a biological phenomenon has taken us on a fascinating journey into chromosome biology, genetic diversity, speciation and evolution. More information on projects relating to parthenogenesis will be available shortly on our home page.

Background on Telomeres
     Telomeres in most eukaryotic cells are comprised of tandem arrays of short G-rich repeats bound by a number of specific proteins. These distinguish chromosome ends from DNA double-strand breaks and prevent the DNA repair machineries from inadvertently joining the natural ends of chromosomes – a mistake that would result in genome instability or cell death. The machinery which replicates the bulk of chromosomal DNA is intrinsically incapable of fully replicating the ends of linear DNA molecules. Due to this 'end-replication problem,' terminal sequences are lost with every round of replication. It is this gradual shortening of chromosomes that led to the view that telomeres act as a “molecular clock” that limits the lifespan of cells and may play a role in organismal aging.

     During early development in stem cells and in most cancer cells, telomeric DNA is replenished by the enzyme telomerase, a ribonucleoprotein complex that reverse transcribes a short region of its RNA subunit to generate new telomeric DNA repeats. Without functional telomerase, chromosomes undergo progressive shortening with each cell division, a process that may act as a natural barrier to cancer as it eventually limits the ability of cells to divide. Most cancer cells have activated telomerase making the enzyme and its regulators attractive targets for anti-cancer drugs. Conversely, some mutations in human telomerase are responsible for a group of degenerative disorders characterized by shortening telomeres and insufficient proliferative capacity of specific cell populations. In recent years, intriguing correlations between telomere length and susceptibility to a variety of diseases have been reported. While the underlying mechanisms are largely unclear, these intriguing findings underscore that much remains to be learned about the structures that cap our chromosomes.

The following is a selection of ongoing studies in our laboratory:

Telomerase biogenesis, recruitment, and activity
     This project is aimed at elucidating the mechanisms by which telomerase is assembled and recruited to chromosome ends and how the enzyme is activated once at telomeres. Difficulties in identifying non-coding RNAs have long hampered the use of S. pombe in studying telomerase regulation as the gene encoding the telomerase RNA subunit (ter1⁺) had not been identified. We recently succeeded in cloning ter1⁺ and have started characterizing the RNA subunit. This work has opened the door for structural and functional analyses of telomerase and provided us with an essential tool for studying telomerase biogenesis and regulation.

Telomere structure and chromosome end protection
     Cytogenetic analysis of irradiated Drosophila cells led Herman Muller to assert that a “terminal gene” seals the ends of all chromosomes and “that (chromosome) fragments die if their ends do not consist of natural termini.” Defining the make-up of these natural termini, or telomeres, has been a matter of considerable interest ever since. Although numerous telomere binding proteins have been identified and characterized, the molecular mechanism that prevents DNA repair factors from acting at chromosome ends remains poorly understood. Widely accepted models involve the presence of large molecular complexes (telomeric heterochromatin) or intramolecular strand invasion resulting in a telomeric loop structure (t-loop). Using a biochemical approach, we have defined the minimal requirements for the protection of telomeric DNA ends from non-homologous end joining (NHEJ), a major double-strand break repair pathway in mammalian cells. Neither long, single-stranded overhangs nor t-loops were required to prevent illegitimate repair of telomeric ends. Instead, a tandem array of 12 telomeric repeats impedes NHEJ in a highly directional manner consistent with the orientation of naturally occurring telomeres. Biochemical fractionation and reconstitution revealed that telomere protection is mediated by a RAP1/TRF2 complex, providing the first demonstration of a direct role for human RAP1 in the protection of telomeric DNA from NHEJ. Mechanistic studies into how cells distinguish chromosome ends from DNA breaks are currently underway. Using assays for homologous pairing and strand invasion, we are also conducting similar experiments to elucidate the effect of telomeric DNA sequences on homologous recombination, the second major DNA repair pathway.

Mechanism of chromosome fusions
     Progressive telomere shortening eventually causes chromosome ends to be recognized as DNA double-strand breaks. This may result in mitotic catastrophe and cell death, but can also lead to bridge-breakage-fusion cycles and chromosomal instability. Fission yeast provides an excellent model for studying the mechanism(s) by which chromosome fusions occur as circularization of all three chromosomes generates a viable product amenable to further analysis. Surprisingly, we found that chromosome circles are generated in the absence of key factors required for DNA double-strand break repair. Using genetic screens and candidate-driven approaches, we have now identified genes that are essential for the formation of circular chromosomes. Data obtained in fission yeast is now guiding studies in human cells to identify factors that promote genome instability in cells with critically short telomeres.

Pot1 - Protection of Telomeres
     As a postdoc with Tom Cech, I identified a fission yeast protein with a critical function in telomere maintenance. Deletion of the encoding gene caused rapid loss of telomeric DNA, a phenotype that prompted me to name the gene Pot1 for Protection of Telomeres. Homologs of Pot1 exist in most eukaryotes, and consistent with a role for Pot1 in protecting chromosome ends, purified human and yeast Pot1 bind to the single-stranded G-rich overhang at the ends of telomeres. More recently, we found that certain mutations in Pot1 cause dramatic telomere lengthening, hence uncovering a role for Pot1 in regulating telomerase recruitment or activity. Our studies suggest that the amount of telomere-bound Pot1 is tightly controlled to regulate access by telomerase but prevent catastrophic loss of telomeres. The regulation of Pot1 at multiple levels is the subject of ongoing studies.

Selected publications

Box JA, Bunch JT, Tang W, Baumann P. Spliceosomal cleavage generates the 3' end of telomerase RNA. Nature. 2008;456:910-914. Abstract

Wang X, Baumann P. Chromosome Fusions following Telomere Loss Are Mediated by Single-Strand Annealing. Mol Cell. 2008;31:463-473. Abstract

Box JA, Bunch JT, Zappulla DC, Glynn EF, Baumann P. A flexible template boundary element in the RNA subunit of fission yeast telomerase. J Biol Chem. 2008;283:24224-24233. Abstract

Leonardi J, Box JA, Bunch JT, Baumann P. TER1, the RNA subunit of fission yeast telomerase. Nat Struct Mol Biol. 2008;15:26-33. Abstract

Bae NS, Baumann P. A RAP1/TRF2 Complex Inhibits Nonhomologous End-Joining at Human Telomeric DNA Ends. Mol Cell. 2007;26:323-334. Abstract

Xhemalce B, Riising EM, Baumann P, Dejean A, Arcangioli B, Seeler JS. Role of SUMO in the dynamics of telomere maintenance in fission yeast. Proc Natl Acad Sci U S A. 2007;104:893-898. Abstract

Baumann P. Are mouse telomeres going to pot? Cell. 2006;126:33-36. Abstract

Baumann P. Taking control of G-quadruplexes. Nat Struct Mol Biol. 2005;12:832-833. Abstract

Bunch JT, Bae NS, Leonardi J, Baumann P. Distinct requirements for pot1 in limiting telomere length and maintaining chromosome stability. Mol Cell Biol. 2005;25:5567-5578. Abstract

Trujillo KM, Bunch JT, Baumann P. Extended DNA binding site in Pot1 broadens sequence specificity to allow recognition of heterogeneous fission yeast telomeres. J Biol Chem. 2005;280:9119-9128. Abstract

Colgin LM, Baran K, Baumann P, Cech TR, Reddel RR. Human POT1 Facilitates Telomere Elongation by Telomerase. Curr Biol. 2003;13:942-946. Abstract

Lei M, Podell ER, Baumann P, Cech TR. DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA. Nature. 2003;426:198-203. Abstract

Lei M, Baumann P, Cech TR. Cooperative Binding of Single-Stranded Telomeric DNA by the Pot1 Protein of Schizosaccharomyces pombe. Biochemistry. 2002;41:14560-14568.  Abstract

Baumann P, Podell E, Cech TR. Human Pot1 (protection of telomeres) protein: cytolocalization, gene structure, and alternative splicing. Mol Cell Biol. 2002;22:8079-8087. Abstract

Baumann P, Cech TR. Pot1, the putative telomere end-binding protein in fission yeast and humans. Science. 2001;292:1171-1175. Abstract

Baumann P, Cech TR. Protection of telomeres by the Ku protein in fission yeast. Mol Biol Cell. 2000;11:3265-3275. Abstract

Haering CH, Nakamura TM, Baumann P, Cech TR. Analysis of telomerase catalytic subunit mutants in vivo and in vitro in Schizosaccharomyces pombe. Proc Natl Acad Sci USA. 2000;97:6367-6372. Abstract