Telomere DNA recognition in Saccharomycotina yeast: potential lessons for the co-evolution of ssDNA and dsDNA-binding proteins and their target sites

In principle, alterations in the telomere repeat sequence would be expected to disrupt the protective nucleoprotein complexes that confer stability to chromosome ends, and hence relatively rare events in evolution. Indeed, numerous organisms in diverse phyla share a canonical 6 bp telomere repeat unit (5′-TTAGGG-3′/5′-CCCTAA-3′), suggesting common descent from an ancestor that carries this particular repeat. All the more remarkable, then, are the extraordinarily divergent telomere sequences that populate the Saccharomycotina subphylum of budding yeast. These sequences are distinguished from the canonical telomere repeat in being long, occasionally degenerate, and frequently non-G/C-rich. Despite the divergent telomere repeat sequences, studies to date indicate that the same families of single-strand and double-strand telomere binding proteins (i.e., the Cdc13 and Rap1 families) are responsible for telomere protection in Saccharomycotina yeast. The recognition mechanisms of the protein family members therefore offer an informative paradigm for understanding the co-evolution of DNA-binding proteins and the cognate target sequences. Existing data suggest three potential, inter-related solutions to the DNA recognition problem: (i) duplication of the recognition protein and functional modification; (ii) combinatorial recognition of target site; and (iii) flexibility of the recognition surfaces of the DNA-binding proteins to adopt alternative conformations. Evidence in support of these solutions and the relevance of these solutions to other DNA-protein regulatory systems are discussed

Combinatorial recognition of a complex telomere repeat sequence by the Candida parapsilosis Cdc13AB heterodimer

The telomere repeat units of Candida species are substantially longer and more complex than those in other organisms, raising interesting questions concerning the recognition mechanisms of telomere-binding proteins. Herein we characterized the properties of Candida parapsilosis Cdc13A and Cdc13B, two paralogs that are responsible for binding and protecting the telomere G-strand tails. We found that Cdc13A and Cdc13B can each form complexes with itself and a heterodimeric complex with each other. However, only the heterodimer exhibits high-affinity and sequence-specific binding to the telomere G-tail. EMSA and crosslinking analysis revealed a combinatorial mechanism of DNA recognition, which entails the A and B subunit making contacts to the 3′ and 5′ region of the repeat unit. While both the DBD and OB4 domain of Cdc13A can bind to the equivalent domain in Cdc13B, only the OB4 complex behaves as a stable heterodimer. The unstable Cdc13ABDBD complex binds G-strand with greatly reduced affinity but the same sequence specificity. Thus the OB4 domains evidently contribute to binding by promoting dimerization of the DBDs. Our investigation reveals a rare example of combinatorial recognition of single-stranded DNA and offers insights into the co-evolution of telomere DNA and cognate binding proteins

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

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

T-OLA analysis of unpaired G-strand overhangs in the wild-type and Δ strains

<p><b>Copyright information:</b></p><p>Taken from "Modulation of telomere terminal structure by telomerase components in "</p><p>Nucleic Acids Research 2006;34(9):2710-2722.</p><p>Published online 19 May 2006</p><p>PMCID:PMC1464115.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () G-strand overhang dynamics was analyzed in successive generations of the wild type and the mutant strain by T-OLA. Selected T-OLA products are designated by the number of repeats that they contain. The strains and passages analyzed in the different parts of the figure are BWP17, streaks 4–10; , streaks 1–9. () The relative levels of G-strand overhangs were quantified for the wild-type (streaks 4, 6, 8 and 10) and samples (streaks 1–9) as described in Materials and Methods, and the averages and deviations plotted

The telomerase ribonucleoprotein in Candida

doi:10.1093/nar/gkl34

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

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

Regulation of Telomere Structure and Functions by Subunits of the INO80 Chromatin Remodeling Complex▿ †

ATP-dependent chromatin remodeling complexes have been implicated in the regulation of transcription, replication, and more recently DNA double-strand break repair. Here we report that the Ies3p subunit of the Saccharomyces cerevisiae INO80 chromatin remodeling complex interacts with a conserved tetratricopeptide repeat domain of the telomerase protein Est1p. Deletion of IES3 and some other subunits of the complex induced telomere elongation and altered telomere position effect. In telomerase-negative mutants, loss of Ies3p delayed the emergence of recombinational survivors and stimulated the formation of extrachromosomal telomeric circles in survivors. Deletion of IES3 also resulted in heightened levels of telomere-telomere fusions in telomerase-deficient strains. In addition, a delay in survivor formation was observed in an Arp8p-deficient mutant. Because Arp8p is required for the chromatin remodeling activity of the INO80 complex, the complex may promote recombinational telomere maintenance by altering chromatin structure. Consistent with this notion, we observed preferential localization of multiple subunits of the INO80 complex to telomeres. Our results reveal novel functions for a subunit of the telomerase complex and the INO80 chromatin remodeling complex

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

Oligonucleotides used in the single-molecule FRET experiments.

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