Probing the mechanism of recognition of ssDNA by the Cdc13-DBD

The Saccharomyces cerevisiae protein Cdc13 tightly and specifically binds the conserved G-rich single-stranded overhang at telomeres and plays an essential role in telomere end-protection and length regulation. The 200 residue DNA-binding domain of Cdc13 (Cdc13-DBD) binds an 11mer single-stranded representative of the yeast telomeric sequence [Tel11, d(GTGTGGGTGTG)] with a 3 pM affinity and specificity for three bases (underlined) at the 5' end. The structure of the Cdc13-DBD bound to Tel11 revealed a large, predominantly aromatic protein interface with several unusual features. The DNA adopts an irregular, extended structure, and the binding interface includes a long ( approximately 30 amino acids) structured loop between strands beta2-beta3 (L(2-3)) of an OB-fold. To investigate the mechanism of ssDNA binding, we studied the free and bound states of Cdc13-DBD using NMR spectroscopy. Chemical shift changes indicate that the basic topology of the domain, including L(2-3), is essentially intact in the free state. Changes in slow and intermediate time scale dynamics, however, occur in L(2-3), while conformational changes distant from the DNA interface suggest an induced fit mechanism for binding in the 'hot spot' for binding affinity and specificity. These data point to an overall binding mechanism well adapted to the heterogeneous nature of yeast telomeres.     

Structural basis for telomeric single-stranded DNA recognition by yeast Cdc13

The essential budding yeast telomere-binding protein Cdc13 is required for telomere replication and end protection. Cdc13 specifically binds telomeric, single-stranded DNA (ssDNA) 3' overhangs with high affinity using an OB-fold domain. We have determined the high-resolution solution structure of the Cdc13 DNA-binding domain (DBD) complexed with a cognate telomeric ssDNA. The ssDNA wraps around one entire face of the Cdc13-DBD OB-fold in an extended, irregular conformation. Recognition of the ssDNA bases occurs primarily through aromatic, basic, and hydrophobic amino acid residues, the majority of which are evolutionarily conserved among budding yeast species and contribute significantly to the energetics of binding. Contacting five of 11 ssDNA nucleotides, the large, ordered beta2-beta3 loop is crucial for complex formation and is a unique elaboration on the binding mode commonly observed in OB-fold proteins. The sequence-specific Cdc13-DBD/ssDNA complex presents a complementary counterpoint to the interactions observed in the Oxytricha nova telomere end-binding and Schizosaccharomyces pombe Pot1 complexes. Analysis of the Cdc13-DBD/ssDNA complex indicates that molecular recognition of extended single-stranded nucleic acids may proceed via a folding-type mechanism rather than resulting from specific patterns of hydrogen bonds. The structure reported here provides a foundation for understanding the mechanism by which Cdc13 recognizes GT-rich heterogeneous sequences with both unusually strong affinity and high specificity.     

Site-directed mutagenesis reveals the thermodynamic requirements for single-stranded DNA recognition by the telomere-binding protein Cdc13

The essential Saccharomyces cerevisiae protein Cdc13 binds the conserved single-stranded overhang at the end of telomeres and mediates access of protein complexes involved in both end-capping and telomerase activity. The single-stranded DNA-binding domain (ssDBD) of Cdc13 exhibits both high affinity (K(d) of 3 pM) and sequence specificity for the GT-rich sequences present at yeast telomeres. We have used the ssDBD of Cdc13 to understand the sequence-specific recognition of extended single-stranded DNA (ssDNA). The recent structure of the Cdc13 DNA-binding domain revealed that ssDNA is recognized by a large protein surface containing an oligonucleotide/oligosaccharide-binding fold (OB-fold) augmented by an extended 30-amino acid loop. Contacts to ssDNA occur via a contiguous surface of aromatic, hydrophobic, and basic residues. A complete alanine scan of the binding interface has been used to determine the contribution of each contacting side chain to binding affinity. Substitution of any aromatic or hydrophobic residue at the interface was deleterious to binding (20 to >700-fold decrease in binding affinity), while tolerance for replacement of basic residues was observed. The important aromatic and hydrophobic contacts are spread throughout the extended interface, indicating that the entire surface is both structurally and thermodynamically required for binding. While all of these contacts are important, several of the individual alanine substitutions that abolish binding cluster to one region of the protein surface. This region is vital for recognition of four bases at the 5' end of the DNA and constitutes a "hotspot" of binding affinity.     

“Poisoning” yeast telomeres distinguishes between redundant telomere capping pathways

In most eukaryotes, telomeres are composed of tandem arrays of species-specific DNA repeats ending with a G-rich 3′ overhang. In budding yeast, Cdc13 binds this overhang and recruits Ten1–Stn1 and the telomerase protein Est1 to protect (cap) and elongate the telomeres, respectively. To dissect and study the various pathways employed to cap and maintain the telomere end, we engineered telomerase to incorporate Tetrahymena telomeric repeats (G₄T₂) onto the telomeres of the budding yeast Kluyveromyces lactis. These heterologous repeats caused telomere–telomere fusions, cell cycle arrest at G2/M, and severely reduced viability—the hallmarks of telomere uncapping. Fusing Cdc13 or Est1 to universal minicircle sequence binding protein (UMSBP), a small protein that binds the single-stranded G₄T₂ repeats, rescued the cell viability and restored telomere capping, but not telomerase-mediated telomere maintenance. Surprisingly, Cdc13–UMSBP-mediated telomere capping was dependent on the homologous recombination factor Rad52, while Est1–UMSBP was not. Thus, our results distinguish between two, redundant, telomere capping pathways.     

Linear chromosome maintenance in the absence of essential telomere-capping proteins

Telomeres were defined by their ability to cap chromosome ends. Proteins with high affinity for the structure at chromosome ends, binding the G-rich, 3' single-stranded overhang at telomeres include Pot1 in humans and fission yeast, TEBP in Oxytricha nova and Cdc13 in budding yeast. Cdc13 is considered essential for telomere capping because budding yeast that lack Cdc13 rapidly accumulate excessive single-stranded DNA (ssDNA) at telomeres, arrest cell division and die. Cdc13 has a separate, critical role in telomerase recruitment to telomeres. Here, we show that neither Cdc13 nor its partner Stn1 are necessary for telomere capping if nuclease activities that are active at uncapped telomeres are attenuated. Recombination-dependent and -independent mechanisms permit maintenance of chromosomes without Cdc13. Our results indicate that the structure of the eukaryotic telomere cap is remarkably flexible and that changes in the DNA damage response allow alternative strategies for telomere capping to evolve.     

Tpz1TPP1 SUMOylation reveals evolutionary conservation of SUMO‐dependent Stn1 telomere association

Elongation of the telomeric overhang by telomerase is counteracted by synthesis of the complementary strand by the CST complex, CTC1(Cdc13)/Stn1/Ten1. Interaction of budding yeast Stn1 with overhang‐binding Cdc13 is increased by Cdc13 SUMOylation. Human and fission yeast CST instead interact with overhang‐binding TPP1/POT1. We show that the fission yeast TPP1 ortholog, Tpz1, is SUMOylated. Tpz1 SUMOylation restricts telomere elongation and promotes Stn1/Ten1 telomere association, and a SUMO‐Tpz1 fusion protein has increased affinity for Stn1. Our data suggest that SUMO inhibits telomerase through stimulation of Stn1/Ten1 action by Tpz1, highlighting the evolutionary conservation of the regulation of CST function by SUMOylation.     

Human POT1 facilitates telomere elongation by telomerase

Mammalian telomeric DNA is mostly composed of double-stranded 5'-TTAGGG-3' repeats and ends with a single-stranded 3' overhang. Telomeric proteins stabilize the telomere by protecting the overhang from degradation or by remodeling the telomere into a T loop structure. Telomerase is a ribonucleoprotein that synthesizes new telomeric DNA. In budding yeast, other proteins, such as Cdc13p, that may help maintain the telomere end by regulating the recruitment or local activity of telomerase have been identified. Pot1 is a single-stranded telomeric DNA binding protein first identified in fission yeast, where it was shown to protect telomeres from degradation [10]. Human POT1 (hPOT1) protein is known to bind specifically to the G-rich telomere strand. We now show that hPOT1 can act as a telomerase-dependent, positive regulator of telomere length. Three splice variants of hPOT1 were overexpressed in a telomerase-positive human cell line. All three variants lengthened telomeres, and splice variant 1 was the most effective. hPOT1 was unable to lengthen the telomeres of telomerase-negative cells unless telomerase activity was induced. These data suggest that a normal function of hPOT1 is to facilitate telomere elongation by telomerase.     

Characterization of the DNA binding features of Saccharomyces castellii Cdc13p

The principal function of Saccharomyces cerevisiae Cdc13p is to provide a loading platform to recruit complexes that provide end protection and telomere replication. We isolated the Saccharomyces castellii Cdc13p homolog (scasCdc13p) and characterized the in vitro DNA binding features of the purified recombinant scasCdc13p. The full-length scasCdc13p binds specifically to G-rich single-stranded telomeric DNA, and not to double-stranded DNA or the C-rich strand. Moreover, the minimal binding site for scasCdc13p is the octamer 5'-GTGTCTGG-3' of the S.castellii telomeric sequence. The scasCdc13p displayed a high affinity binding, where four individual nucleotide residues were found to be of most importance for the sequence specificity. Nonetheless, scasCdc13p binds the telomeric repeats from various other species, including the human. In spite of considerable divergence in telomere repeat length and sequence between these species, a conserved Cdc13p binding motif was detected. Among the budding yeasts this conserved Cdc13p binding site overlaps the Rap1p binding site. Together, these data implicate scasCdc13p as a telomere end-binding protein with a potential role in the regulation of telomere maintenance in vivo. Moreover, the results suggest that Rap1p and Cdc13p act together to preserve the conserved core present within the otherwise highly divergent btelomeric sequences among a wide variety of yeasts.     

Specific binding of single-stranded telomeric DNA by Cdc13p of Saccharomyces cerevisiae

Cdc13p is a single strand telomere-binding protein of Saccharomyces cerevisiae; its telomere-binding region is within amino acids 451-693, Cdc13(451-693)p. In this study, we used purified Cdc13p and Cdc13(451-693)p to characterize their telomere binding activity. We found that the binding specificity of single-stranded TG(1-3) DNA by these two proteins is similar. However, the affinity of Cdc13(451-693)p to DNA was slightly lower than that of Cdc13p. The binding of telomeric DNA by these two proteins was disrupted at NaCl concentrations higher than 0.3 m, indicating that electrostatic interaction contributed significantly to the binding process. Because both proteins bound to strand TG(1-3) DNA positioned at the 3' end, the 5' end, or in the middle of the oligonucleotide substrates, our results indicated that the location of TG(1-3) in single-stranded DNA does not appear to be important for Cdc13p binding. Moreover, using DNase I footprint analysis, the structure of the telomeric DNA complexes of Cdc13p and Cdc13(451-693)p was analyzed. The DNase I footprints of these two proteins to three different telomeric DNA substrates were virtually identical, indicating that the telomere contact region of Cdc13p is within Cdc13(451-693)p. Together, the binding properties of Cdc13p and its binding domain support the theory that the specific binding of Cdc13p to telomeres is an important feature of telomeres that regulate telomerase access and/or differentiate natural telomeres from broken ends.     

Cdc13 cooperates with the yeast Ku proteins and Stn1 to regulate telomerase recruitment

The Saccharomyces cerevisiae CDC13 protein binds single-strand telomeric DNA. Here we report the isolation of new mutant alleles of CDC13 that confer either abnormal telomere lengthening or telomere shortening. This deregulation not only depended on telomerase (Est2/TLC1) and Est1, a direct regulator of telomerase, but also on the yeast Ku proteins, yKu70/Hdf1 and yKu80/Hdf2, that have been previously implicated in DNA repair and telomere maintenance. Expression of a Cdc13-yKu70 fusion protein resulted in telomere elongation, similar to that produced by a Cdc13-Est1 fusion, thus suggesting that yKu70 might promote Cdc13-mediated telomerase recruitment. We also demonstrate that Stn1 is an inhibitor of telomerase recruitment by Cdc13, based both on STN1 overexpression and Cdc13-Stn1 fusion experiments. We propose that accurate regulation of telomerase recruitment by Cdc13 results from a coordinated balance between positive control by yKu70 and negative control by Stn1. Our results represent the first evidence of a direct control of the telomerase-loading function of Cdc13 by a double-strand telomeric DNA-binding complex.     

Est1 protects telomeres and inhibits subtelomeric y'-element recombination

In the budding yeast Saccharomyces cerevisiae, the structure and function of telomeres are maintained by binding proteins, such as Cdc13-Stn1-Ten1 (CST), Yku, and the telomerase complex. Like CST and Yku, telomerase also plays a role in telomere protection or capping. Unlike CST and Yku, however, the underlying molecular mechanism of telomerase-mediated telomere protection remains unclear. In this study, we employed both the CDC13-EST1 fusion gene and the separation-of-function allele est1-D514A to elucidate that Est1 provided a telomere protection pathway that was independent of both the CST and Yku pathways. Est1's ability to convert single-stranded telomeric DNA into a G quadruplex was required for telomerase-mediated telomere protection function. Additionally, Est1 maintained the integrity of telomeres by suppressing the recombination of subtelomeric Y' elements. Our results demonstrate that one major functional role that Est1 brings to the telomerase complex is the capping or protection of telomeres.     

Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p

Appropriate control of the chromosome end-replicating enzyme telomerase is crucial for maintaining telomere length and genomic stability. The essential telomeric DNA-binding protein Cdc13p both positively and negatively regulates telomere length in budding yeast. Here we test the effect of purified Cdc13p on telomerase action in vitro. We show that the full-length protein and its DNA-binding domain (DBD) inhibit primer extension by telomerase. This inhibition occurs by competitive blocking of telomerase access to DNA. To further understand the requirements for productive telomerase 3′-end access when Cdc13p or the DBD is bound to a telomerase substrate, we constrained protein binding at various distances from the 3′-end on two sets of increasingly longer oligonucleotides. We find that Cdc13p inhibits the action of telomerase through three distinct biochemical modes, including inhibiting telomerase even when a significant tail is available, representing a novel ‘action at a distance’ inhibitory activity. Thus, while yeast Cdc13p exhibits the same general activity as human POT1, providing an off switch for telomerase when bound near the 3′-end, there are significant mechanistic differences in the ways telomere end-binding proteins inhibit telomerase action.     

Mutually exclusive binding of telomerase RNA and DNA by Ku alters telomerase recruitment model

In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes Ku's telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt nonhomologous end-joining (NHEJ), telomeric gene silencing, and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both in?vitro and in?vivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. PAPERCLIP:     

Interaction of an Arabidopsis RNA-binding protein with plant single-stranded telomeric DNA modulates telomerase activity

Telomeres are the specialized structures at the end of linear chromosomes and terminate with a single-stranded 3' overhang of the G-rich strand. The primary role of telomeres is to protect chromosome ends from recombination and fusion and from being recognized as broken DNA ends. This protective function can be achieved through association with specific telomere-binding proteins. Although proteins that bind single-stranded G-rich overhang regulate telomere length and telomerase activity in mammals and lower eukaryotes, equivalent factors have yet to be identified in plants. Here we have identified proteins capable of interacting with the G-rich single-stranded telomeric repeat from the Arabidopsis extracts by affinity chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis indicates that the isolated protein is a chloroplast RNA-binding protein (and a truncated derivative). The truncated derivative, which we refer to as STEP1 (single-stranded telomere-binding protein 1), binds specifically the single-stranded G-rich plant telomeric DNA sequences but not double-stranded telomeric DNA. Unlike the chloroplast-localized full-length RNA-binding protein, STEP1 localizes exclusively to the nucleus, suggesting that it plays a role in plant telomere biogenesis. We also demonstrated that the specific binding of STEP1 to single-stranded telomeric DNA inhibits telomerase-mediated telomere extension. The evidence presented here suggests that STEP1 is a telomere-end binding protein that may contribute to telomere length regulation by capping the ends of chromosomes and thereby repressing telomerase activity in plants.     

Prediction of multiple tandem OB-fold domains in telomere end-binding proteins Pot1 and Cdc13

The heterodimeric Oxytricha nova telomere end binding protein, the original telomere end binding protein characterized, contains four OB-fold domains used for recognition of single-stranded telomeric DNA. In contrast, only solitary OB-fold domains have been found in the telomere end binding proteins from yeast and higher eukaryotes. Using a sliding-window algorithm coupled with sequence profile-profile analysis, we provide support for the existence of multiple OB-fold domains in two other telomeric ssDNA binding proteins, vertebrate Pot1 and budding yeast Cdc13. This common usage of multiple, tandem OB-fold domains in telomeric end binding proteins extends the known evolutionary conservation of eukaryotic end-protection mechanisms.     

Exonuclease activity is required for sequence addition and Cdc13p loading at a de novo telomere.

The Saccharomyces cerevisiae Mre11p/Rad50p/Xrs2p (MRX) complex is evolutionarily conserved and functions in DNA repair and at telomeres [1-3]. In vivo, MRX is required for a 5' --> 3' exonuclease activity that mediates DNA recombination at double-strand breaks (DSBs). Paradoxically, abolition of this exonuclease activity in MRX mutants results in shortened telomeric DNA tracts. To further explore the role of MRX at telomeres, we analyzed MRX mutants in a de novo telomere addition assay in yeast cells [4]. We found that the MRX genes were absolutely required for telomerase-mediated addition in this assay. Furthermore, we found that Cdc13p, a single-stranded telomeric DNA binding protein essential for telomere DNA synthesis and protection [5], was unable to bind to the de novo telomeric DNA substrate in cells lacking Rad50p. Based on the results from this model system, we propose that the MRX complex helps to prepare telomeric DNA for the loading of Cdc13p, which then protects the chromosome from further degradation and recruits telomerase and other DNA replication components to synthesize telomeric DNA.     

Sequence-specific binding to telomeric DNA is not a conserved property of the Cdc13 DNA binding domain

In the budding yeast Saccharomyces cerevisiae, chromosome end protection is provided by a heterotrimeric complex composed of Cdc13 in association with the RPA-like proteins Stn1 and Ten1. We report here that the high affinity and specificity of the S. cerevisiae Cdc13 DNA binding domain for single-stranded telomeric DNA are not widely shared by other fungal Cdc13 proteins, suggesting that restriction of this complex to telomeres may be limited to the Saccharomyces clade. We propose that the evolutionarily conserved task of Stn1 and Ten1 (and their associated large subunit) is a genome-wide role in DNA replication rather than a telomere-dedicated activity.     

Delivery of yeast telomerase to a DNA break depends on the recruitment functions of Cdc13 and Est1

The yeast single-strand TG-repeat telomere binding protein Cdc13 and the telomerase accessory protein Est1 play essential roles in chromosome end replication. To determine whether a proposed Cdc13-Est1 interaction recruits telomerase (Est2), we used a simplified system in which telomere formation was monitored at an HO-induced DNA double-strand break (DSB). Tethering of either Cdc13 or Est1 adjacent to a DSB promoted telomere formation, and tethering of Est1, even in the absence of a DSB, resulted in the recruitment of Est2. Est1 association with a DSB containing an adjacent short TG-repeat sequence depended on the Cdc13-Est1 interaction affected by cdc13-2 and est1-60 mutations, whereas Cdc13 association did not. Similarly, Est2 binding to the DSB also required the Cdc13-Est1 interaction, but not synthesis of new TG repeats at the break site. These data demonstrate a critical role for Est1 in recruiting telomerase to its site of action, in cooperation with the telomere binding protein Cdc13.     

Species-specific and sequence-specific recognition of the dG-rich strand of telomeres by yeast telomerase.

A gel mobility shift assay was developed to examine recognition of yeast telomeres by telomerase. An RNase-sensitive G-rich strand-specific binding activity can be detected in partially purified yeast telomerase fractions. The binding activity was attributed to telomerase, because it co-purifies with TLC1 RNA and telomerase activity over three different chromatographic steps and because the complex co-migrates with TLC1 RNA when subjected to electrophoresis through native gels. Analysis of the binding specificity of yeast telomerase indicates that it recognizes the G-rich strand of yeast telomeres with high affinity and specificity. The K d for the interaction is approximately 3 nM. Single-stranded G-rich telomeres from other species, such as human and Tetrahymena, though capable of being extended by yeast telomerase in polymerization assays at high concentrations, bind the enzyme with at least 100-fold lower affinities. The ability of a sequence to be bound tightly by yeast telomerase in vitro correlates with its ability to seed telomere formation in vivo. The implications of these findings for regulation of telomerase activity are discussed.     

Identification and characterization of a putative telomere end-binding protein from Tetrahymena thermophila.

Telomeric DNA of Tetrahymena thermophila consists of a long stretch of (TTGGGG)n double-stranded repeats with a single-stranded (TTGGGG)2 3' overhang at the end of the chromosome. We have identified and characterized a protein that specifically binds to a synthetic telomeric substrate consisting of duplex DNA and the 3' telomeric repeat overhang. This protein is called TEP (telomere end-binding protein). A change from G to A in the third position of the TTGGGG overhang repeat converts the substrate to a human telomere analog and reduces the binding affinity approximately threefold. Changing two G's to C's in the TTGGGG repeats totally abolishes binding. However, permutation of the Tetrahymena repeat sequence has only a minor effect on binding. A duplex structure adjacent to the 3' overhang is required for binding, although the duplex need not contain telomeric repeats. TEP does not bind to G-quartet DNA, which is formed by many G-rich sequences. TEP has a greatly reduced affinity for RNA substrates. The copy number of TEP is at least 2 x 10(4) per cell, and it is present under different conditions of cell growth and development, although its level varies. UV cross-linking experiments show that TEP has an apparent molecular mass of approximately 65 kDa. Unlike other telomere end-binding proteins, TEP is sensitive to high salt concentrations.