Telomeric repeat-binding factor 1 (Tbf1) has a similar architecture as the TRF family of telomeric proteins and plays important roles in both telomere homeostasis and ribosome regulation. However, the molecular basis of why Tbf1 has such different functions compared to other TRFs remains unclear. Here, we present the crystal structures of the TRF homology (TRFH) and Myb-L domains from Schizosaccharomyces pombe Tbf1 (spTbf1). TRFH-mediated homodimerization is essential for spTbf1 stability. Importantly, spTbf1_TRFH lacks the conserved docking motif for interactions with telomeric proteins, explaining why spTbf1 does not participate in the assembly of the shelterin complex. Finally, structural and biochemical analyses demonstrate that TRFH and Myb-L domains as well as the loop region of spTbf1 coordinate to recognize S. pombe telomeric double-stranded DNA. Overall, our findings provide structural and functional insights into how fungi Tbf1 acts as an atypical telomeric repeat-binding factor, which helps to understand the evolution of TRFH-containing telomeric proteins.

端粒重复结合因子 1 (Tbf1) 与端粒蛋白 TRF 家族具有相似的结构，在端粒稳态和核糖体调节中发挥重要作用。然而，Tbf1 与其他 TRF 相比具有如此不同功能的分子基础仍不清楚。在这里，我们展示了裂殖酵母Tbf1 (spTbf1)的 TRF 同源 (TRFH) 和 Myb-L 结构域的晶体结构。 TRFH 介导的同二聚化对于 spTbf1 的稳定性至关重要。重要的是，spTbf1 _TRFH缺乏与端粒蛋白相互作用的保守对接基序，这解释了为什么 spTbf1 不参与庇护蛋白复合物的组装。最后，结构和生化分析表明，TRFH 和 Myb-L 结构域以及 spTbf1 的环区域协调识别粟酒裂殖酵母端粒双链 DNA。总体而言，我们的研究结果为真菌 Tbf1 如何作为非典型端粒重复结合因子发挥作用提供了结构和功能见解，这有助于了解含有 TRFH 的端粒蛋白的进化。