The role of shelterin components on the structural dynamics of telomeric DNA

Telomeres are the nucleoprotein caps found at chromosome ends. They help to differentiate the chromosome ends from DNA double strand breaks and define the molecular clock of a cell. The molecular clock determines the number of times a cell can divide and when telomeres significantly shorten, cellular senescence or growth arrest occurs. Cancer cells reverse this process either by activating telomerase or by utilizing Alternative Lengthening of Telomeres (ALT). Suppression of ALT, regulation of telomerase, and end capping are all performed by the shelterin complex. The shelterin complex is composed of six proteins; TRF1, TRF2, Rap1, TIN2, TPP1 and Pot1. Rap1, TRF2 and Pot1 are important for the suppression of ALT and regulate the topology of the telomeric DNA. Telomeric DNA is composed of G-rich tandem repeats and this unique character facilitates the formation of higher order structures such as t-loops and G-quadruplexes. Even though both t-loops and G-quadruplexes are important for end protection, they need to be resolved for replication/transcription complexes to progress. Presented here is an investigation into how Rap1, Pot1 and their associated complexes TRF2/Rap1 and Pot1/TPP1 mediate structural changes of telomeric DNA and whether t-circles could act as a substrate for the telomere extension through ALT. We observed that hRap1 directly interacts with DNA and modulates the localization of TRF2 at telomeric ends by increasing its affinity and specificity to telomeric sequences and telomeric junction sites. As a result, the TRF2/Rap1 complex can form more t-loops than TRF2 alone, presenting one possible mechanism of end protection and explanation of hRap1's role. We also demonstrated that G-rich ss telomeric DNA exists as a beads-on-a-string conformation with each bead size ~500 nucleotides. Furthermore, we showed that Pot1 and the Pot1/TPP1 complex can form filaments along G-rich ss telomeric DNA as they open up the G-quadruplexes. These findings can explain how the telomeric DNA structure is modulated during replication. Additionally, we showed that only C-rich t-circles can act as a substrate for telomere extension by a rolling circle mechanism. These information all together demonstrate the importance of Rap1, TRF2 and Pot1 and can be used to explain how ALT initiates upon loss of the shelterin components.Doctor of Philosoph