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.

MTV, an ssDNA Protecting Complex Essential for Transposon-Based Telomere Maintenance in Drosophila

“…Therefore, even though CST complexes share similar biological functions in different organisms, their modes of interaction and even complex composition can be remarkably diverse. For this latter point, the stoichiometry of CST in Candida was recently determined to be 2:4:2 or 2:6:2 rather than 1:1:1 [30]. Therefore, proving a complex is structurally analogous to CST is not trivial in any system, let alone in Drosophila where the protein divergence seems to be at the extreme.…”

Dynamic Interactions of Arabidopsis TEN1: Stabilizing Telomeres in Response to Heat Stress

“…In the absence of heat shock, AtTEN1 migrated as a discrete band between 232 and 140 kD on native PAGE ( Figure 5B). Because human and yeast CST complexes exhibit an apparent molecular weight of $500 kD in SEC (Lue et al, 2013;Miyake et al, 2009), we suspect this AtTEN1-containing complex does not represent a trimeric Arabidopsis CST. Strikingly, under the same heat shock conditions that destabilized AtCTC1, (D) AtSTN1 does not protect CTC1 from heat-induced aggregation.…”

Duplication and Functional Specialization of the Telomere-capping Protein Cdc13 in Candida Species

“…Note that the fraction of probe in the complexes represents less than 1% of the total probe used in these assays. activity of Cdc13A (13). His 6 -SUMO-Stn1 was expressed alone or co-expressed with GST-Cdc13B-FLAG in E. coli (Fig.…”