Purification and characterization of the <i>C. glabrata</i> CST complex.

<p>(A) The fusion proteins co-expressed in <i>E. coli</i> are schematically illustrated. (B) Extracts were isolated from <i>E. coli</i> strains co-expressing Stn1 and Ten1 or all three subunits, and subjected sequentially to Ni-NTA and M2 affinity chromatography. The extracts and eluates from the Ni-NTA and M2 resins were analyzed by SDS-PAGE and Coomassie staining. (C) The M2 eluate containing the CST complex from part B was subjected to Glutathione-Sepharose affinity chromatography. The flow through (FT) and eluate fractions as well as the starting material were analyzed by SDS-PAGE and Coomassie staining. (D) Different amounts of the purified CST complex (10 µl, 5 µl and 2.5 µl) and bovine serum albumin (5 µg, 2.5 µg, 1.25 µg, 0.63 µg, and 0.32 µg) were analyzed by SDS-PAGE and Coomassie staining. (E) Serial three fold dilutions of the purified CST complex were subjected to Western analysis using antibodies directed against the SUMO tag, which is present in both the Cdc13 and Stn1 fusion proteins. (F) Purified SUMO-Cdc13 and the CST complex were separately fractionated on 15–30% glycerol gradients. The distributions of the proteins in the fractions were analyzed by Western using antibodies directed against SUMO (the Cdc13 and Stn1 fusions) and GST (the Ten1 fusion protein). The results for free Cdc13 and the CST complex are displayed in the top panel and the bottom three panels, respectively.</p

Crosslinking of <i>C. glabrata</i> Stn1 and Ten1 to photo-reactive telomere oligonucleotides.

<p>(A) Purified protein complex containing SUMO-Stn1N and GST-Ten1 fusion, as well as GST-Ten1 alone, was analyzed by SDS-PAGE and Coomassie staining. (B) Increasing concentrations of the indicated Stn1-Ten1 complexes (6.6 nM, 20 nM, 60 nM) were crosslinked to labeled IdU-1 or IdU-5 oligonucleotides and the covalent products detected by SDS-PAGE and PhosphorImager scanning. (C) Increasing concentrations of the Stn1-Ten1 complex or GST-Ten1 alone (6.6 nM, 20 nM, 60 nM) were crosslinked to labeled IdU-1 or IdU-5 oligonucleotides and the covalent products detected by SDS-PAGE and PhosphorImager scanning. (D) Purified <i>Cg</i>Stn1-Ten1 complex (240 nM) was crosslinked to labeled IdU-1 oligonucleotide (16 nM) in the presence of increasing concentrations of the indicated competitor oligonucleotides (125 nM, 500 nM, 2 µM and 8 µM).</p

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

<div><p>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 <em>Candida glabrata</em> 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 <em>Candida albicans</em> Stn1 OB fold domain caused a reduction in its crosslinking efficiency <em>in vitro</em> and engendered long and heterogeneous telomeres <em>in vivo</em>, 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.</p> </div

Oligonucleotides used in the single-molecule FRET experiments.

<p>Oligonucleotides used in the single-molecule FRET experiments.</p

Cdc13 and CST form unstable complexes with non-telomeric DNA.

<p>(A) The sequences of the non-telomeric oligonucleotides used in the gel mobility shift assays are displayed. (B) Increasing concentrations of purified Cdc13 (0 nM, 40 nM and 80 nM) were incubated with the indicated oligonucleotide probes (7.5 nM) and the resulting complexes analyzed by gel electrophoresis and PhosphorImager scanning. (C) Labeled R1 and R3 probes (7.5 nM) were incubated with BSA (80 and 240 nM), Cdc13 (40 nM), and CST (40 nM), and the resulting complexes analyzed by gel electrophoresis and PhosphorImager scanning.</p

Cdc13 and CST can bind and unfold higher order G-tail structure.

<p>(A) A schematic depiction of reaction steps for single molecule FRET experiments. (B) Single molecule FRET efficiency histograms in the presence of the indicated concentrations of Cdc13. (C) Single molecule FRET efficiency histograms in the presence of the indicated concentrations of CST. (D–F) Representative single-molecule FRET-time traces of the 48-nt G-tail construct in 3 mM MgCl₂ and 100 mM NaCl before adding any protein. A 532 nm laser was used for excitation. (G) A representative single-molecule FRET-time trace of the 48-nt G-tail construct in 3 mM MgCl₂ and 100 mM NaCl immediately after adding 1 nM CST, showing a CST binding event at ∼19 sec (the black arrow). A 532 nm laser was used for Cy3 excitation throughout the entire course of data acquisition. A 633 nm laser was briefly turned on for direct Cy5 excitation at ∼86 sec in order to confirm that Cy5 is still active rather than photobleached.</p

Stn1, but not Ten1, independently binds to Cdc13.

<p>(A) <i>E. coli</i> extracts prepared from strains co-expressing SUMO-Cdc13-FG and HIS₆-SUMO-Stn1 (CS) or SUMO-Cdc13-FG and GST-Ten1 (CT) were subjected to Ni-NTA, Glutathione-Sepharose and M2 affinity purification as indicated. The extracts and eluates were analyzed by SDS-PAGE and Coomassie staining (top), as well as Western using antibodies directed against the FLAG tag, the HIS₆ tag and the GST tag of Cdc13, Stn1 and Ten1 fusion protein, respectively (bottom). Significant amounts of Cdc13 proteolytic fragments (*) can be detected in the CT eluate from the M2 resin. (B) <i>E. coli</i> extracts prepared from strains expressing HIS₆-SUMO-Stn1 alone (S) or HIS₆-SUMO-Stn1 and SUMO-Cdc13-FG (CS) were subjected to M2 affinity purification. The Cdc13 and Stn1 fusion proteins in the input and eluate samples were detected by Western using antibodies directed against the FLAG and HIS₆ tag, respectively. (C) <i>E. coli</i> extracts prepared from strains expressing GST-Ten1 alone (T) or GST-Ten1 and SUMO-Cdc13-FG (CT) were subjected to M2 affinity purification. The Cdc13 and Ten1 fusion proteins in the input and eluate samples were detected by Western using antibodies directed against the FLAG and GST tag, respectively.</p