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

端粒末端序列的高效复制对于维持端粒稳定性至关重要。在裂殖酵母中，Taz1蛋白作为TRF1-2的同源蛋白，在端粒复制过程中发挥核心作用。我们与其他研究团队均已证实，Stn1-Ten1（ST）复合物在端粒与亚端粒的维持中发挥重要功能，但其具体作用机制尚未完全阐明。本研究采用聚合酶使用测序（polymerase usage sequencing, PuSeq）分析全基因组复制动态，该技术可实现全基因组范围内聚合酶使用情况的评估。我们的研究结果显示，ST复合物与Taz1蛋白对于STE2-3亚端粒区域的复制至关重要。ST复合物与Taz1蛋白均可特异性结合该区域，且ST复合物的功能依赖于其与shelterin复合物的相互作用。值得注意的是，当ST复合物功能受损时，基于同源重组（Homologous Recombination, HR）的复制叉重启机制便成为保障STE2-3区域复制的必要途径。最后，我们证实，亚端粒区域内受Rif1调控的复制起始位点的激活，可有效规避亚端粒的复制缺陷。综上，我们证实shelterin复合物可主动将ST复合物招募至亚端粒区域，从而保障具备难复制区域典型特征的STE2-3区域的复制。实验整体设计：我们以单核苷酸分辨率定位了突变型DNA聚合酶δ（DNA polymerase delta, Polδ）与ε（DNA polymerase epsilon, Polε）所掺入的核糖核苷酸，并通过后续生物信息学分析解析全基因组复制动态。