Tpz1-Ccq1 and Tpz1-Poz1 Interactions within Fission Yeast Shelterin Modulate Ccq1 Thr93 Phosphorylation and Telomerase Recruitment

Abstract

In both fission yeast and humans, the shelterin complex plays central roles in regulation of telomerase recruitment, protection of telomeres against DNA damage response factors, and formation of heterochromatin at telomeres. While shelterin is essential for limiting activation of the DNA damage checkpoint kinases ATR and ATM at telomeres, these kinases are required for stable maintenance of telomeres. In fission yeast, Rad3 ^ATR and Tel1 ^ATM kinases are redundantly required for telomerase recruitment, since Rad3 ^ATR/Tel1 ^ATM-dependent phosphorylation of the shelterin subunit Ccq1 at Thr93 promotes interaction between Ccq1 and the telomerase subunit Est1. However, it remained unclear how protein-protein interactions within the shelterin complex (consisting of Taz1, Rap1, Poz1, Tpz1, Pot1 and Ccq1) contribute to the regulation of Ccq1 Thr93 phosphorylation and telomerase recruitment. In this study, we identify domains and amino acid residues that are critical for mediating Tpz1-Ccq1 and Tpz1-Poz1 interaction within the fission yeast shelterin complex. Using separation of function Tpz1 mutants that maintain Tpz1-Pot1 interaction but specifically disrupt either Tpz1-Ccq1 or Tpz1-Poz1 interaction, we then establish that Tpz1-Ccq1 interaction promotes Ccq1 Thr93 phosphorylation, telomerase recruitment, checkpoint inhibition and telomeric heterochromatin formation. Furthermore, we demonstrate that Tpz1-Poz1 interaction promotes telomere association of Poz1, and loss of Poz1 from telomeres leads to increases in Ccq1 Thr93 phosphorylation and telomerase recruitment, and telomeric heterochromatin formation defect. In addition, our studies establish that Tpz1-Poz1 and Tpz1-Ccq1 interactions redundantly fulfill the essential telomere protection function of the shelterin complex, since simultaneous loss of both interactions caused immediate loss of cell viability for the majority of cells and generation of survivors with circular chromosomes. Based on these findings, we suggest that the negative regulatory function of Tpz1-Poz1 interaction works upstream of Rad3 ^ATR kinase, while Tpz1-Ccq1 interaction works downstream of Rad3 ^ATR kinase to facilitate Ccq1 Thr93 phosphorylation and telomerase recruitment.

Author Summary

Proper maintenance of telomeres is essential for maintaining genomic stability, and genomic instability caused by dysfunctional telomeres could lead to accumulation of mutations that may drive tumor formation. Telomere dysfunction has also been linked to premature aging caused by depletion of stem cells. Therefore, it is important to understand how cells ensure proper maintenance of telomeres. Mammalian cells and fission yeast cells utilize an evolutionarily conserved multi-subunit telomere protection complex called shelterin to ensure protection against telomere fusions by DNA repair factors and cell cycle arrest by DNA damage checkpoint kinases. However, previous studies have not yet fully established how protein-protein interactions within the shelterin complex contribute to the regulation of DNA damage checkpoint signaling and telomerase recruitment. By utilizing separation of function mutations that specifically disrupt either Tpz1-Ccq1 or Tpz1-Poz1 interaction within the fission yeast shelterin, we establish that Tpz1-Ccq1 interaction is essential for phosphorylation of Ccq1 by the DNA damage checkpoint kinases Rad3 ^ATR and Tel1 ^ATM that is needed for telomerase recruitment to telomeres, while Tpz1-Poz1 interaction prevents Ccq1 phosphorylation by promoting Poz1 association with telomeres. These findings thus establish for the first time how protein-protein interactions within the shelterin complex modulate checkpoint kinase-dependent phosphorylation essential for telomerase recruitment.