title: 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.
authors.affiliation: Department of Microbiology and Immunology, W. R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, New York, New York, USA. nflue@med.cornell.edu
authors.forename: Neal F
authors.initials: NF
authors.lastname: Lue
authors.forename: Ruobo
authors.initials: R
authors.lastname: Zhou
authors.forename: Lidia
authors.initials: L
authors.lastname: Chico
authors.forename: Ninghui
authors.initials: N
authors.lastname: Mao
authors.forename: Olga
authors.initials: O
authors.lastname: Steinberg-Neifach
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
mesh_terms: D002176:Candida albicans / Q000235:genetics / Q000378:metabolism; D041221:Candida glabrata / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D016615:Telomere / Q000235:genetics* / Q000378:metabolism; D034501:Telomere-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: C452219:Cdc13 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; C428109:Stn1 protein, S cerevisiae; C512860:TEN1 protein, S cerevisiae; D034501:Telomere-Binding Proteins