We are interested in understanding how defects in telomere maintenance lead to genome instability. Towards this aim, my laboratory employs a variety of techniques in biochemistry, molecular genetics, and cell biology to dissect and elucidate the functions of the protein-nucleic acid complexes found at the ends of chromosomes.  As chromosome end protection and telomere maintenance are common challenges for all organisms with linear chromosomes, several of our projects rely on the fission yeast Schizosaccharomyces pombe as a model system. 

Our research has been guided by two fundamental questions:

(I) How are chromosome ends distinguished from DNA double strand breaks?

(II) What happens when end protection fails?

(I) 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 has been a matter of considerable interest for many scientists ever since. Although numerous telomere binding proteins have been identified and characterized, the molecular mechanisms that prevent DNA repair factors from acting at chromosome ends have remained elusive. Widely accepted models involve the presence of large molecular complexes (telomeric heterochromatin) or intramolecular strand invasion generating telomeric loop structures. 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 DNA double-strand break repair pathway in mammalian cells (Bae and Baumann, 2007). Neither long, single-stranded overhangs nor t-loops are  required to prevent illegitimate repair of telomeric ends in an in vitro end joining assay. 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 TRF2/hRAP1 complex, providing the first evidence for a direct role for human RAP1 in the protection of telomeric DNA from NHEJ (Figure 1).

Current efforts are aimed at defining the molecular mechanism by which hRAP1 blocks the NHEJ machinery. Using assays for homologous pairing and strand invasion we will conduct similar experiments to elucidate the effect of telomeric DNA sequences and telomere binding proteins on homologous recombination, the second major repair pathway for DNA double strand breaks.  Further complementing the in vitro biochemistry are RNAi screens for novel proteins involved in chromosome end protection and fusion of uncapped telomeres.

(II) Mechanism of chromosome fusions

Progressive telomere shortening eventually causes chromosome ends to be recognized as DNA double-strand breaks.  Mechanistic insights into chromosome end fusions have predominantly come from experiments in which the telomeric chromatin structure was disrupted by loss of a telomere binding protein.  For example, when fission yeast cells lacking the double-stranded telomeric DNA binding protein Taz1 are arrested in the G1 phase of the cell cycle, chromosomal end fusions ensue. These fusions retain telomeric DNA and are dependent on ligase 4 and Ku, key components of the non-homologous end joining (NHEJ) pathway. Similarly, removal of the mammalian Taz1 homolog TRF2 from human or mouse telomeres results in NHEJ-mediated telomere fusions.

Fusions between the ends of different chromosomes produce unstable dicentric products.  In contrast, a fusion between the two ends of one chromosome generates a circular chromosome, a relatively stable product that has been observed in cells from a variety of organisms including humans.  The fact that the haploid fission yeast genome is distributed over only three chromosomes favors intrachromosomal fusions in this organism compared to most others with higher chromosome number.  When fission yeast cells fail to maintain telomeres, for example following loss of the telomere end binding protein Pot1, inter- and intrachromosomal fusions can be observed (Baumann and Cech). While cells harboring interchromosomal fusions suffer severe chromosome segregation defects, cells with three circular chromosomes quickly emerge as stable survivors that are amenable to further analysis.  Taking advantage of this phenomenon, we have used genetic screens and candidate-driven approaches to elucidate the repair processes responsible for telomere attrition-induced chromosome fusions.  Intriguingly, chromosome circularization was not compromised in strains deleted for key components of the NHEJ or classic recombination pathways, indicating that the striking NHEJ–dependence of chromosome fusions following loss of Taz1/TRF2 is not representative of fusions triggered by telomere erosion. Instead, we found that telomere attrition-induced chromosome fusions are dependent on the fission yeast homologs of Rad52, the ERCC1/XPF endonuclease, the single-stranded DNA binding protein RPA, and the Srs2 and Werner/Bloom helicases (Wang and Baumann, 2008).  These factors define the single-strand annealing pathway. 

Further experiments in our lab demonstrated that the circumstances and cause of telomere dysfunction profoundly affect which DNA repair pathway is engaged at denuded chromosome ends (Figure 2). We are now building on these results by investigating the mechanism of chromosome fusions triggered by various insults to telomeres.  Future studies in fission yeast will focus on the molecular characterization of the different pathways that act at chromosome ends and on the mechanism by which the cause of uncapping affects which DNA repair pathway is engaged. We have also started to apply what we have learnt from fission yeast to human cells and data obtained in fission yeast will be guiding our efforts to identify mammalian factors that promote genome instability in cells with compromised telomere function.