A Sequential Assembly Mechanism for Stable Cdc13 Dimerization on Telomeric DNA

The telomere-binding protein Cdc13 specifically binds to single-stranded telomeric DNA, playing a critical role in telomere protection and length regulation. While extensive biochemical, molecular biological, and genetic studies have shown that Cdc13 can form dimers or oligomers in solution and bind telomeric DNA with high specificity, the dynamic mechanism of its loading onto telomeres is less well characterized. Combining two single-molecule methods, single-molecule fluorescence resonance energy transfer (smFRET) and colocalization single-molecule spectroscopy (CoSMoS), we demonstrate a kinetically favored sequential assembly pathway on a 12-nucleotide telomeric DNA segment involving an initial Cdc13 binding event followed by recruitment of a second Cdc13 molecule. While genetic studies suggest that monomeric Cdc13 binding alone is insufficient to maintain telomere length, our findings underscore the Cdc13 monomer’s regulatory importance in coordinating telomere synthesis and protection. This monomer-to-dimer transition provides a mechanistic basis for understanding the multifunctional roles of Cdc13 in telomere replication and protection.