Fission Yeast Shelterin Regulates DNA Polymerases and Rad3 ATR Kinase to Limit Telomere Extension

摘要

Studies in fission yeast have previously identified evolutionarily conserved shelterin and Stn1-Ten1 complexes, and established Rad3~(ATR)/Tel1~(ATM)-dependent phosphorylation of the shelterin subunit Ccq1 at Thr93 as the critical post-translational modification for telomerase recruitment to telomeres. Furthermore, shelterin subunits Poz1, Rap1 and Taz1 have been identified as negative regulators of Thr93 phosphorylation and telomerase recruitment. However, it remained unclear how telomere maintenance is dynamically regulated during the cell cycle. Thus, we investigated how loss of Poz1, Rap1 and Taz1 affects cell cycle regulation of Ccq1 Thr93 phosphorylation and telomere association of telomerase (Trt1~(TERT)), DNA polymerases, Replication Protein A (RPA) complex, Rad3~(ATR)-Rad26~(ATRIP)checkpoint kinase complex, Tel1~(ATM)kinase, shelterin subunits (Tpz1, Ccq1 and Poz1) and Stn1. We further investigated how telomere shortening, caused by trt1Δ or catalytically dead Trt1-D743A, affects cell cycle-regulated telomere association of telomerase and DNA polymerases. These analyses established that fission yeast shelterin maintains telomere length homeostasis by coordinating the differential arrival of leading (Polε) and lagging (Polα) strand DNA polymerases at telomeres to modulate Rad3~(ATR)association, Ccq1 Thr93 phosphorylation and telomerase recruitment. Author Summary Stable maintenance of telomeres is critical to maintain a stable genome and to prevent accumulation of undesired mutations that may lead to formation of tumors. Telomere dysfunction can also lead to premature aging due to depletion of the stem cell population, highlighting the importance of understanding the regulatory mechanisms that ensure stable telomere maintenance. Based on careful analysis of cell cycle-regulated changes in telomere association of telomerase, DNA polymerases, Replication Protein A, checkpoint kinases, telomere protection complex shelterin, and Stn1-Ten1 complex, we will provide here a new and dynamic model of telomere length regulation in fission yeast, which suggests that shelterin-dependent regulation of differential arrival of leading and lagging strand DNA polymerase at telomeres is responsible for modulating Rad3~(ATR)checkpoint kinase accumulation and Rad3~(ATR)-dependent phosphorylation of shelterin subunit Ccq1 to control telomerase recruitment to telomeres.