Molecular insights into assembly of the yeast CST-Polα/primase complex for telomere maintenance

Telomeric DNA replication involves coordinated action of telomerase and DNA polymerase α-primase (Polα/primase), which synthesize the G-strand and C-strand, respectively. The conserved Cdc13-Stn1-Ten1 (CST) complex critically regulates this process by both terminating telomerase activity and stimulating Polα/primase. While telomerase-mediated G-overhang synthesis is well studied, how CST-Polα/primase is recruited to telomeres for C-strand fill-in remains poorly understood. Structural analysis reveals an evolutionary diversity of CST interaction with Polα/primase across organisms. Here, using a combination of AlphaFold3 structural modeling with genetic assays, we unveil the assembly mechanism of CST-Polα/primase for telomere maintenance in Saccharomyces cerevisiae . We structurally characterize the uniqueness and specific determinants of the Stn1-Pol12 interaction in the S. cerevisiae CST-Polα/primase complex. The Cdc13-Pol1 and Stn1-Pol12 interactions together mediate CST-dependent recruitment of Polα/primase to telomeres. Functional analyses reveal that disruption of the CST-Polα/primase interaction shows distinct telomere phenotypes compared to CST disassembly, supporting the model that CST recruits Polα/primase after telomerase elongation is terminated. Together, our findings not only resolve the longstanding question on the assembly mechanism of the CST and Polα/primase complexes at yeast telomeres, but also hint that CST-Polα/primase-dependent C-strand fill-in likely governs recombination-based telomere structure maintenance via controlling 3’ overhang length.