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The telomere capping complex CST has an unusual stoichiometry, makes multipartite interaction with G-Tails, and unfolds higher-order G-tail structures.

TitleThe telomere capping complex CST has an unusual stoichiometry, makes multipartite interaction with G-Tails, and unfolds higher-order G-tail structures.
Publication TypeJournal Article
Year of Publication2013
AuthorsLue NF, Zhou R, Chico L, Mao N, Steinberg-Neifach O, Ha T
JournalPLoS Genet
Volume9
Issue1
Paginatione1003145
Date Published2013
ISSN1553-7404
KeywordsCandida albicans, Candida glabrata, Cell Cycle Proteins, Chromosomal Proteins, Non-Histone, DNA Replication, Fungal Proteins, Protein Binding, Protein Folding, Protein Structure, Tertiary, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Telomere, Telomere-Binding Proteins
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.

DOI10.1371/journal.pgen.1003145
Alternate JournalPLoS Genet
PubMed ID23300477
PubMed Central IDPMC3536697
Grant ListR01 GM065367 / GM / NIGMS NIH HHS / United States
GM062631 / GM / NIGMS NIH HHS / United States
GM065367 / GM / NIGMS NIH HHS / United States
/ / Howard Hughes Medical Institute / United States

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