Telomeres are specialized nucleoprotein structures at the ends of eukaryotic chromosomes that are essential for chromosome stability and cellular proliferation. Telomeric DNA does not encode for proteins, instead it consists of tandem repeats of TG-rich sequences of double-stranded DNA that terminate in a 3' single-stranded DNA overhang. Protection of this overhang is essential. When left unprotected, this overhang initiates DNA damage responses that lead to catastrophic events permanently damaging the genome and resulting in apoptosis or senescence. Furthermore, telomere shortening due to the inability of the DNA-replication machinery to fully replicate the ends is a critical mechanism of tumor suppression as well as a hallmark of aging. Continually proliferating cells maintain adequate telomeres through the action of the reverse transcriptase telomerase. Telomeres are important to human health because dysregulation of either telomere protection or telomerase activity causes many human diseases. Notably, over 90% of human cancers activate telomerase for continued proliferation.
Our research in this area aims to understand how telomere-associated proteins protect and maintain telomeres. Key questions include how subunits of the telomerase enzyme contribute to activity, how the single-strand DNA overhang is shielded from the DNA-damage machinery, and whether capping activity also regulates telomerase action. We develop this knowledge by first understanding the core activities of key telomere factors, then testing these activities in a reconstituted telomerase assay and validating our knowledge directly in the organism.
The tRPA (CST) Complex
The telomere provides a protective cap for the linear chomosome ends. In S. cerevisiae this function is mediated by Cdc13, and two other proteins, Stn1 and Ten1. Several lines of evidence suggest that these proteins form a putative complex (tRPA/CST), and deletion of any one of these three proteins leads to loss of cellular viability.
Structural characterization of individual domains in the tRPA complex has revealed an organization strikingly similar to the RPA complex. Despite these similarities, tRPA has many telomere specific functions and our lab is working to elucidate the mechanisms behind these functions. We are using both biochemical and biophysical based assays to probe the protein/protein and protein/nucleic acid interactions that occur by tRPA proteins. These experiments will be invaluable in deciphering the broad biochemical functions of both the tRPA complex and telomere proteins in general.
Maintaining proper telomere length is essential for cell survival, thus telomerase is carefully controlled by both positive and negative regulation. These regulatory mechanisms ensure that the shortest telomeres are preferentially elongated by telomerase, and, conversely, that telomerase is repressed at longer telomeres to prevent over-elongation. The budding yeast telomerase holoenzyme consists of four entities – the catalytic subunit Est2, the associated RNA component TLC1, and two additional subunits Est1 and Est3, which both participate in telomere length regulation. In the absence of any one of these critical factors, telomeres continually shorten as cells divide until replicative senescence is triggered, giving rise the Est name - Ever Shorter Telomeres. The telomerase complex is assembled on the TLC1 RNA scaffold, which has a central core augmented by long, flexible arms. The catalytic subunit Est2 binds the central core, which also contains the essential RNA templating sequence. In a manner independent of Est2, Est1 binds the RNA through a specific interaction with an internal loop at the end of one of these flexible arms. In contrast, Est3’s holoenzyme association depends on the presence of Est2, suggesting a direct protein/protein interaction.
Our lab is engaged in understanding the structures and activities of the components of the yeast core holoenzyme and how the interplay of these activities regulates the action of telomerase. Most recently, we have used new strategies, merging NMR data and ROSETTA, to solve the first structure of a yeast telomerase component, the Est3 protein.