Regulation of Telomere Maintenance by the Shelterin Complex in Fission Yeast Schizosaccharomyces pombe
thesisposted on 2016-06-21, 00:00 authored by Ya-Ting Chang
Telomere replication is tightly controlled during cell cycle progression, especially in late S/G2 phase when 3’-overhang of telomere is extended by telomerase, a specialized reverse transcriptase crucial for compensating DNA loss from the incomplete duplication of telomeric DNA by replicative DNA polymerases. Studies in fission yeast have previously identified evolutionarily conserved shelterin and Stn1-Ten1 complexes, and established that Rad3ATR/Tel1ATM-dependent phosphorylation of the shelterin complex subunit Ccq1 at Thr93 as a critical post-translational modification for telomerase recruitment to telomeres. Furthermore, the shelterin complex subunits Poz1, Rap1 and Taz1 have been identified as negative regulators of Ccq1 Thr93 phosphorylation and telomerase recruitment. However, it remained unclear how telomere maintenance is dynamically regulated during the cell cycle. In the first part of my study, I therefore investigated how loss of Poz1, Rap1 and Taz1 affects cell cycle regulation of Ccq1 Thr93 phosphorylation and telomere association of telomerase (Trt1TERT), DNA polymerases, Replication Protein A (RPA) complex, Rad3ATR-Rad26ATRIP checkpoint kinase complex, Tel1ATM kinase, shelterin subunits (Tpz1, Ccq1 and Poz1) and Stn1. Furthermore, I examined how telomere shortening, caused by trt1 deletion or catalytically dead trt1-D743A, affects cell cycle-regulated telomere association of telomerase and DNA polymerases. Based on these findings, I then proposed that fission yeast shelterin maintains telomere length homeostasis by coordinating the differential arrival of leading (Polε) and lagging (Polalpha) strand DNA polymerases at telomeres to modulate Rad3ATR association, Ccq1 Thr93 phosphorylation and telomerase recruitment. In the second part of my study, I determined that Ccq1-interacting protein, Tpz1 is hyper-phosphorylated by Rad3ATR/Tel1ATM kinases in strains with highly elongated telomeres due to lack of telomerase inhibitors (poz1 deletion, rap1 deletion, and taz1 deletion) as well as in strains with shorter telomeres due to a defect in telomerase mediated telomere extension by telomerase (trt1 deletion and ccq1-T93A). The increase in Tpz1-telomerase interactions mirrored the increase in Tpz1 hyper-phosphorylation observed in poz1 deletion, rap1 deletion and taz1 deletion cells, raising the possibility that Tpz1 phosphorylation might be important in promoting telomerase-shelterin interaction and telomerase mediated telomere extension. Furthermore, deletion of the ccq1 gene abolished Tpz1 hyper-phosphorylation, suggesting that Ccq1 functions as an adaptor to mediate Rad3ATR/Tel1ATM-dependent phosphorylation of Tpz1. However, abolishment of all six high consensus Rad3ATR/Tel1ATM phosphorylation sites with the preferred SQ/TQ motif did not completely eliminate Tpz1 hyper-phosphorylation in rap1 deletion cells nor did it lead to any defect in telomere length maintenance, suggesting that non-SQ/TQ sites within Tpz1 must be phosphorylated. Therefore, to more comprehensively determine Tpz1 phosphorylation sites and examine functional significance of Tpz1 phosphorylation in telomere maintenance, I identified in vivo Tpz1 phosphorylation sites by mass spectrometry analysis and carried out mutational analysis of these sites. My analysis revealed that a non-Rad3ATR/ Tel1ATM phosphorylation site Ser138 negatively regulated telomere extension, while multiple phosphorylation sites within a central S/T rich region of Tpz1 positively regulated both telomere extension and phosphorylation-dependent mobility shift of Tpz1 observed in rap1 deletion cells. These findings thus established that phosphorylation of Tpz1 contributes to telomere length homeostasis.