Structural And Functional Studies Of The Human Telomeric Pot1-Tpp1 Complex And Its Role In Human Disease
Telomeres are essential regions of repetitive DNA at the ends of eukaryotic chromosomes that serve an indispensable role in the protection and replication of the genome. Telomeres shorten over time due to nucleolytic degradation, oxidative damage, and through a process known as the ‘end replication problem,’ which shortens telomeres after every round of genome replication. Critically short telomeres are linked to cellular senescence, aging, and human disease. Therefore, it is imperative to understand the molecular mechanisms that regulate and protect telomere length. Correct regulation of telomere length is key and is facilitated by two telomere capping complexes: shelterin and CST. The human shelterin complex is composed of TRF1, TRF2, Rap1, TIN2, POT1, and TPP1, binds single and double stranded telomeric DNA and protects telomeres from degradation and unwanted DNA repair. The human CST complex, composed of CTC1, Stn1, and Ten1, bind and caps the single-stranded overhang of telomeres. These two complexes also maintain telomere length by regulating access of telomerase to telomeres, the reverse transcriptase that extends the length of telomeres. In humans, the shelterin subcomplex, POT1-TPP1, is thought to directly engage and enhance the processivity of telomerase. Conversely, CTC1 of the human CST complex interacts with TPP1 inhibiting the activity of telomerase at telomeres. Furthermore, the CST complex is involved in the recruitment of Pol to telomeres; the DNA polymerase involved in the replication of the telomeric C-strand. As a result, the interaction between the shelterin and CST complexes represents a potential key mechanism for telomere length regulation that is still not fully understood. Naturally occurring mutations in patients with rare aging disorders and cancers have been recently linked to telomere capping complexes. Elucidating the structural and functional consequences of these naturally occurring mutations will allow us to better understand how dysfunctional telomere maintenance results in human disease. My dissertation focuses on the functional and structural data of the human POT1-TPP1 complex, which reveals that naturally occurring mutations in the C-terminal domain of POT1 result in partial disruption of the POT1-TPP1 complex and telomere phenotypes associated with unregulated telomere length.