The Telomere Capping Complex CST Has an Unusual Stoichiometry, Makes Multipartite Interaction with G-Tails, and Unfolds Higher-Order G-Tail Structures

Abstract

The telomere-ending binding protein complex CST (Cdc13-Stn1-Ten1) mediates critical functions in both telomere protection and replication. We devised a co-expression and affinity purification strategy for isolating large quantities of the complete Candida glabrata CST complex. The complex was found to exhibit a 2∶4∶2 or 2∶6∶2 stoichiometry as judged by the ratio of the subunits and the native size of the complex. Stn1, but not Ten1 alone, can directly and stably interact with Cdc13. In gel mobility shift assays, both Cdc13 and CST manifested high-affinity and sequence-specific binding to the cognate telomeric repeats. Single molecule FRET-based analysis indicates that Cdc13 and CST can bind and unfold higher order G-tail structures. The protein and the complex can also interact with non-telomeric DNA in the absence of high-affinity target sites. Comparison of the DNA–protein complexes formed by Cdc13 and CST suggests that the latter can occupy a longer DNA target site and that Stn1 and Ten1 may contact DNA directly in the full CST–DNA assembly. Both Stn1 and Ten1 can be cross-linked to photo-reactive telomeric DNA. Mutating residues on the putative DNA–binding surface of Candida albicans Stn1 OB fold domain caused a reduction in its crosslinking efficiency in vitro and engendered long and heterogeneous telomeres in vivo, indicating that the DNA–binding activity of Stn1 is required for telomere protection. Our data provide insights on the assembly and mechanisms of CST, and our robust reconstitution system will facilitate future biochemical analysis of this important complex.

Author Summary

Telomeres are the special structures that protect the chromosomal ends against aberrant rearrangements. The most distal portion of a telomere is bound by the CST (Cdc13-Stn1-Ten1) complex, which is critical for telomere protection. By preparing and analyzing large quantities of the CST complex derived from a fungus called Candida glabrata, we showed that CST has an intricate arrangement and each complex probably contains 2, 4 (or 6), and 2 copies of the Cdc13, Stn1, and Ten1 protein, respectively. By investigating the interaction between pairs of subunits, we provide support for the notion that Stn1 acts as a bridge to bring the three components together. In addition, using a technique that measures the properties of individual fluorescent DNA molecules, we demonstrated that telomere single-stranded DNA has a tendency to fold back on itself, but that the binding of the CST complex to DNA disrupts the folding and makes the DNA more extended. We also showed that Stn1 can touch DNA directly and that this property is required for Stn1 to protect telomeres in the cell. Finally, we found that CST can bind DNA that carries non-telomeric sequences, suggesting the complex may play a role at other chromosomal locations.