The shelterin-dependent recruitment of Stn1-Ten1 ensures replication of hard to replicate subtelomeric regions in fission yeast

Efficient replication of terminal sequences is crucial to maintain telomere stability. In fission yeast, the Taz1 protein, the TRF1-2 homolog, plays a prominent role in telomere replication. We and others have shown that the Stn1-Ten1 (ST) complex fulfills important function in telomeres and subtelomeres maintenance, however its function is not fully understood. Here, we analyzed genome-wide replication dynamics using the polymerase usage sequencing (PuSeq) allowing to assess polymerase usage across the genome. We show that the ST complex and Taz1 are crucial for the replication of the STE2-3 subtelomeric region. Both, the ST complex and Taz1, bind specifically to this region and the function of the ST complex relies on its association with the shelterin complex. Strikingly, when the ST complex is compromised, an HR-based fork restart mechanism becomes necessary to ensure replication of the STE2-3 region. Finally, we demonstrate that the firing of an origin, that is controlled by Rif1, within subtelomeric regions circumvents the replication defect of subtelomeres. Overall, we demonstrate that the ST complex is actively recruited at subtelomeres by the shelterin and this ensures replication of the STE2-3 region which exhibits specific features of a hard-to-replicate region. We mapped ribonucleotide-incorporation by the mutated DNA polymerase delta and epsilon at single-nucleotide resolution and used the data for following replication dynamics by subsequent informatics analysis.