Rif1 acts through Protein Phosphatase 1 and independent of replication timing to suppresses telomere extension in budding yeast

The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast S. cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant that cannot interact with PP1 causes a long-telomere phenotype, similar to that of rif1∆ cells. Compromised PP1 function also causes telomere extension. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1-PP1 interaction leads to precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if nearby replication origins are deleted, indicating that effects of Rif1 on telomere length are not mediated through replication timing. Instead we find that, even at a telomere created after DNA synthesis during a mitotic block, Rif1-PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.在酿酒酵母（S. cerevisiae）中，Rif1蛋白可负调控端粒TG重复序列的长度，但其阻止端粒过度延伸的具体机制尚未明确。近期研究证实，Rif1可通过充当蛋白磷酸酶1（PP1）的靶向亚基来调控DNA复制过程。据此，我们探究了Rif1是否通过靶向PP1的活性来实现端粒长度的调控。实验结果显示，无法与PP1结合的Rif1突变体可引发端粒过长表型，该表型与rif1∆细胞的端粒异常表型一致。PP1功能受损同样会导致端粒延长。将PP1锚定至特定端粒，可在限制TG重复序列长度方面部分替代Rif1的功能，这一结果证实了PP1在端粒长度调控中的关键作用。破坏Rif1与PP1的相互作用，会导致端粒邻近的复制起点过早激活，同时引发端粒复制异常提前。但我们发现，即便删除了邻近的复制起点，Rif1仍可有效限制端粒长度，这表明Rif1对端粒长度的调控并非通过复制时序途径介导。进一步研究表明，即便在有丝分裂阻滞期间DNA合成后新形成的端粒中，Rif1与PP1的相互作用仍是抑制端粒延长、阻止Tel1激酶异常招募的必要条件。综上，本研究结果证实，Rif1通过招募PP1直接抑制端粒酶介导的TG重复序列延长，从而实现对端粒长度的调控。