Fission yeast RPA–TERT–Tpz1 TPP1 complex promotes telomere extension and suppresses telomere recombination

Abstract Telomerase maintains chromosome ends by extending telomeric DNA, yet how recruited telomerase becomes productively engaged remains poorly understood. Recent studies showed that Replication Protein A (RPA) stimulates telomerase in humans and budding yeast through interactions with TERT and TPP1 orthologs, suggesting a direct role in activation. Here, we provide genetic and structural modeling evidence for a ternary RPA-Trt1TERT-Tpz1TPP1 complex that promotes telomere extension while suppressing recombination in fission yeast. Guided by results from genetic screen, followed by AlphaFold3 modeling and systematic mutagenesis of RPA, Trt1, and Tpz1, we identify four key interfaces supporting telomerase function: Ssb1RPA1-Trt1, Ssb2RPA2-Trt1, Ssb2RPA2-Tpz1, and the TEL-patch-mediated Trt1-Tpz1 interaction. Notably, Tpz1-R81, previously assigned as the TEL patch, instead contacts Ssb2 in the complex. Epistasis and suppressor analyses indicate distinct contributions of the RPA-Trt1 and RPA-Tpz1 interfaces to telomerase activation. Comparative analysis using AlphaFold3 further suggests that these interactions are likely conserved in budding yeast and humans. Together, these findings support a model in which RPA serves as an architectural component that coordinates TERT and TPP1-like factors to enable productive telomerase engagement. Author Summary Telomeres are specialized structures at chromosome ends that must be maintained to preserve genome stability. Telomerase extends telomeric DNA, but how it becomes fully active after being recruited to telomeres remains poorly understood. In this study, we use fission yeast to examine the role of the conserved single-stranded DNA-binding protein Replication Protein A (RPA) in this process. We find that RPA forms a functional complex with the telomerase catalytic subunit (TERT) and the shelterin protein Tpz1 (a homolog of human TPP1). Genetic and structural analyses identify multiple interactions within this complex that are required for efficient telomere extension. Disrupting these interactions allows telomerase to be recruited but prevents productive telomere elongation. Our results also suggest that similar mechanisms may operate in other organisms, including budding yeast and humans. These findings provide insight into how telomerase activity is regulated at chromosome ends. Competing Interest Statement The authors have declared no competing interest.