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Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protectionMing Lei, Elaine R Podell & Thomas R Cech

The POT1 (protection of telomeres 1) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryotes. It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere length in human cells. Here, we report the crystal structure at a resolution of 1.73 Å of the N-terminal half of human POT1 (hPOT1) protein bound to a telomeric single-stranded DNA (ssDNA) decamer, TTAGGGTTAG, the minimum tight-binding sequence indicated by in vitro binding assays. The structure reveals that hPOT1 contains two oligonucleotide/ oligosaccharide-binding (OB) folds; the N-terminal OB fold binds the first six nucleotides, resembling the structure of the S. pombe Pot1pN–ssDNA complex, whereas the second OB fold binds and protects the 3′ end of the ssDNA. These results provide an atomic-resolution model for chromosome end-capping.

Eukaryotic genome stability relies on the presence of telomeres, special-ized protein–DNA complexes that compose the very ends of the linear chromosomes1. Telomeres form protective caps at the ends of chromo-somes that protect them from fusing with each other or with damaged-induced DNA double-strand breaks, and as a result they prevent natural chromosome ends from signaling and initiating DNA damage checkpoint cascades2,3. Finally, telomeres serve as the substrate for the ribonucleo-protein enzyme telomerase, which adds telomeric DNA to the 3′ ends of chromosomes to ensure complete genome replication4. Failures in these functions have been shown to promote the genomic instability associated with cancerous cells, as well as to limit the proliferation of both cancer-ous and normal cells, indicating that telomeres are important players in cellular aging and cancer5–8.

In vertebrates, telomeric DNA comprises noncoding tandem repeats of the sequence 5′-TTAGGG-3′ (ref. 9). Although telomere sequence and length vary from species to species, the protrusion of the G-rich strand as a 3′ single-stranded overhang is conserved among ciliated protozoa, yeast and mammals10–13. The G-rich overhang is the substrate for both telomerase and telomeric ssDNA-binding proteins, which are critical for chromosome end-capping. These proteins include TEBP (telomere end-binding protein) from the hypotrichous ciliates14,15, Cdc13p from budding yeast16,17, and the Pot1 proteins, which are pres-ent in organisms including fission yeast, plants and humans13,18. In the budding yeast Saccharomyces cerevisiae, Cdc13p specifically binds telomeric G-rich overhangs and is essential for telomerase recruitment and/or activation at the telomere19,20. Loss of Cdc13p function leads to resectioning of the C-rich telomeric strand, exposing the G-rich overhang and eliciting a Rad9-dependent cell cycle arrest21. In the

fission yeast S. pombe, Pot1 is essential for the protection of chromo-some ends13 and exhibits high specificity for telomeric ssDNA in vitro22. Deletion of the pot1 gene leads to rapid loss of telomere sequences, chromosome mis-segregation, and chromosome circularization, diag-nostic of chromosome end-to-end fusion13.

Human POT1 protein was identified based on its sequence simi-larity to the Oxytricha nova TEBP α subunit (TEBPα), the first well-characterized telomeric ssDNA-binding protein13. Although the crystal structure of TEBPα revealed three OB folds15, two involved in DNA binding and the third necessary for a protein interaction with TEBPβ, the sequence similarity between hPOT1 and TEBPα is restricted to their N-terminal OB folds. In human cells, hPOT1 localizes to telomeres, and loss of the G-rich overhang causes a reduc-tion in hPOT1 binding18,23. In one study, overexpression of a mutant form of hPOT1 missing the N-terminal OB fold led to rapid telomere lengthening in telomerase-positive cells, whereas overexpression of full-length hPOT1 had no effect on telomere length23. In other stud-ies, however, expression of full-length hPOT1 caused substantial telo-mere elongation by telomerase24,25. Given that the distantly related protein in S. cerevisiae, Cdc13p, can exert both a negative and positive influence on telomere length regulation16, these disparate conclusions might indicate that hPOT1 plays an essential but complicated role in the regulation of telomere length.

Here we present the crystal structure of the DNA-binding domain of hPOT1 in complex with its cognate telomeric ssDNA at a resolution of 1.73 Å. The structure, in conjunction with biochemical assays, provides insights into the mechanism by which hPOT1 recognizes, binds and protects telomeric DNA. Furthermore, we found that the most stable

Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA. Correspondence should be addressed to T.R.C. (thomas.cech@colorado.edu).

Published online 21 November 2004; doi:10.1038/nsmb867

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hPOT1 complex formed with sequences ending not with TTAGGG but with GGTTAG, exactly the sequence synthesized by each round of telomerase action; this observation has implications for telomerase regulation by hPOT1.

RESULTShPOT1 binds a ten-base telomeric ssDNATwo forms of hPOT1 were studied: version 1 (V1), the full-length pro-tein, and version 2 (V2), one of five splicing variants identified previ-ously by mRNA analysis18. The hPOT1V2 mRNA is expressed only in some human tissues and at levels considerably lower than that of the mRNA for the full-length protein. In tissue culture cells, only version 1 has been detected at the protein level23. Thus it is safest to consider V2 as a biochemical construct suitable for studying DNA-protein interactions, although the biological occurrence of this form of the hPOT1 protein remains unconfirmed.

Full-length hPOT1V1 and splicing variant 2 were expressed using a baculovirus-insect cell system and purified (Fig. 1a). Both proteins were used in the following biochemical studies and gave indistinguish-able results (Supplementary Fig. 1 online). Therefore, for simplicity, we hereafter use hPOT1 to represent both hPOT1V1 and hPOT1V2.

We first tested whether hPOT1 could bind to any of the ssDNAs representing the six possible permutations of a single telomeric repeat. At very high protein concentration ([hPOT1] = 450 nM), only TTAGGG showed a limited ability to bind hPOT1 by gel mobility shift assays (Kd >> 450 nM) (Fig. 1b). The other five permuted versions formed no stable complex (Fig. 1b). Thus hPOT1 binds ssDNA very differently from S. pombe Pot1pN, which binds the S. pombe telomeric hexanucleotide GGTTAC with high affinity and specificity22.

Efficient binding to hPOT1 required a telomeric ssDNA of ten bases or longer that included the core telomeric sequence TTAGGGTTAG (hT10)

(Fig. 1c). None of the other permutations of hT10 gave strong binding (10′ in Fig. 1c, and data not shown). This 3′-end sequence requirement agrees with that recently reported by others for human and chicken POT1 (refs. 26,27), but our minimum binding sequence has one more 5′ nucleotide; the other studies used ssDNAs with adjacent 5′ nonte-lomeric sequence, and we find that these 5′ nucleotides contribute to binding (Supplementary Fig. 2 online).

To identify nucleotides critical for stable interaction with hPOT1, a series of telomeric ssDNAs containing single-nucleotide substitutions was evaluated using a gel mobility shift assay (Fig. 1d). The substitu-tions of the two guanine residues at the 5′ end of the ssDNA 12-mer had no effect on binding, confirming that these two positions are not part of the minimum binding sequence. T2, A3, G4 and G5 contributed greatly to protein binding, a result reminiscent of the S. pombe Pot1pN–ssDNA complex, where sequence specificity was greatest for nucleotides 2–5 of the hexanucleotide. The 3′-terminal G10 was also critical (Fig. 1d). Substitutions at T1, G6, T7 and T8 resulted in intermediate affinity for hPOT1, indicating that these positions confer some degree of sequence specificity. Substitution of A9 did not interfere with binding.

A titration experiment at high hT10 concentration provided an accurate value for the concentration of active protein (data not shown). To determine the equilibrium dissociation constant (Kd) of the hPOT1–hT10 complex, the binding of a trace amount of hT10 with increasing hPOT1 was measured. hPOT1 bound to hT10 in a simple binding equilibrium. The stoichiometry of the complex obtained from the abscissa intercept corresponded to one hPOT1 molecule bound per hT10; analysis of the complex by gel filtration confirmed that it consists of a 1:1 protein–DNA complex in solution (data not shown). The Kd for the hPOT1–hT10 complex was 9.2 nM.

To quantify the 3′-end specificity of the Pot1pN-ssDNA interaction, the dissociation constants of hPOT1 in complex with (GGTTAG)2 (GTTAGG)2 and (TTAGGG)2 were measured by the gel shift assay. One extra guanine at the 3′ end did not interfere with the interaction between hPOT1 and the ssDNA (Kd = 10 nM versus 9.4 nM) (Fig. 1e). However, two extra guanines at the 3′ end caused an eight-fold reduction in affinity (71 nM). In contrast, extra nucleotides at the 5′ end had no effect on binding (Supplementary Fig. 3 online).

Figure 1 Characterization of the interaction of hPOT1 with telomeric ssDNA. (a) Two splicing variants of hPOT1. Left, organization of hPOT1V1 and hPOT1V2 polypeptide chains. Residue numbers at the boundaries of various subdivisions are indicated. The two OB folds are labeled. The C-terminal regions of hPOT1V1 and hPOT1V2, which differ in sequence, are gray and black bars, respectively. Right, purified hPOT1V1 and hPOT1V2 were resolved by SDS-PAGE and stained with Coomassie brilliant blue. (b) One telomeric repeat is not sufficient for efficient binding to hPOT1. hPOT1 (500 nM) was incubated with all six possible permutations of a single telomeric repeat (17 nM) as indicated. Complexes were then analyzed by native PAGE. (c) hPOT1 binds to a minimum ten-base telomeric ssDNA, TTAGGGTTAG. The sequences of the telomeric ssDNAs are above the gel. hPOT1 (85 nM) was incubated with the telomeric ssDNAs (17 nM) and then analyzed by native PAGE. (d) Telomeric ssDNA binding specificity of hPOT1. Experimental conditions were the same as in c. One nucleotide at a time was substituted with its complement. The wild-type nucleotides of the 12 positions are labeled. Numerals indicate nucleotide position within the ten-base minimum tight-binding sequence. (e) Determination of the Kd values of three telomeric ssDNAs binding to hPOT1. The telomeric ssDNAs were incubated with the indicated concentrations of hPOT1 under the conditions described in Methods. The solid, dotted and dashed lines represent theoretical curves with Kd[(GGTTAG)2] = 9.5 nM; Kd[(GTTAGG)2] = 10 nM; and Kd[(TTAGGG)2] = 59 nM, respectively. for each experiment (b–e), the other POT1 version was also tested and gave indistinguishable results (Supplementary Fig. 1 online).

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Overall structure of the hPOT1–ssDNA complexTo understand at the molecular level how hPOT1 recognizes its cog-nate telomeric ssDNA, we reconstituted and crystallized the complex of hPOT1V2 and hT10. We determined the crystal structure at a resolution of 1.73 Å by MAD using both a mercury compound (MetHgAc) and a synthetic ssDNA with 5-bromouracil incorporated (Table 1). The final model contains residues 5–145 and 149–299 of hPOT1V2, and has been refined to an R-value of 22.8% (Rfree = 23.7%). Electron density for the entire DNA was observed (Fig. 2a).

The crystal structure shows that hPOT1V2 adopts an elongated con-formation (Fig. 2b) and is composed of two OB fold28,29 domains closely connected by a short linker (Fig. 2c). The OB fold is a five-stranded β-barrel present in many prokaryotic and eukaryotic ssDNA-binding proteins15,17,30–32. Previous amino acid sequence comparisons detected only the first OB fold of hPOT1. Failure to detect OB2 may be due to a large insertion between β2 and β3 (residues 186–217) of OB2. Notably, the existence of the second OB fold has recently been predicted using a new sequence-profile analysis33. The ssDNA-binding domain of Cdc13 (Cdc13-DBD) also has a large insertion between β2 and β3 (30 resi-dues)17,34. OB1 (residues 5–140) and OB2 (residues 149–299) pack in tandem, with their individual ssDNA-binding grooves connected, to create a single continuous channel with a kink at the OB1-OB2 interface. This arrangement is different from that seen in the RPA–ssDNA30 and the BRCA2–ssDNA35 crystal structures, in which the ssDNA-binding grooves in the two OB folds are oriented in the same direction.

The protein-ssDNA interactionThe telomeric ssDNA in the complex spans OB1 and OB2, binding in the continuous basic concave groove (Fig. 2b). The DNA adopts an irregular and extended conformation with its backbone exposed to solvent and its bases partially or completely buried in a total solvent-excluded contact area of 900 Å2. T1–G5 interact exclusively with OB1, whereas T7–G10 interact with OB2. The phosphate group preceding G6 interacts with both OB folds. There is a sharp kink in the ssDNA backbone at the phosphodiester group of T7, where the ssDNA crosses between the two OB folds; this causes the long axis of the DNA to deviate from its initial direction by ∼90°.

The conformation of the ssDNA is governed by four sets of pairwise stacking interactions: T1 stacks with T2, A3 with G4, G5 with G6, and T8 with A9; one side of each stack packs face-to-face against an aromatic amino acid side chain (Fig. 2d). The same sort of interac-tion occurs three times in the S. pombe Pot1pN–ssDNA complex32.

Although the base of T7 does not stack with other nucleotides, it is sandwiched in an extended stack of aromatic side chains (Fig. 2d). The base of G10 stacks against the side chain of Tyr223 and, on the other face, packs against the backbone of A9. All of these stacking interac-tions between bases and aromatic residues presumably contribute to binding affinity but not to sequence specificity.

OB1 makes much more extensive contact with the ssDNA than does OB2. There are a total of 22 hydrogen bonds between T1-G6 and the residues of OB1, over two-thirds of those (31 in total) observed in the complex (Fig. 3). Thus T1-G6 would be expected to be the major binding site for hPOT1, consistent with the in vitro binding data (Fig. 1b). Among these hydrogen bonds, 13 are sequence-specific and involve Watson-Crick donor-acceptor groups and protein residues located on the surface of the binding groove. OB2 makes nine hydrogen bonds with the ssDNA, eight of which are contributed by T7 and G10. A9 is the only nucleotide in the ssDNA whose base does not make any hydrogen bonding interaction, suggesting that position 9 should not be sequence-specific. This observa-tion is consistent with the result of the binding assay (Fig. 1d).

The bases of A3 and G6 make hydrogen bonding interactions with the phosphodiester groups of the previous nucleotides T2 and G5, respec-tively. The same DNA ‘self-recognition’ interaction also occurs with the base of A5 in the structure of the S. pombe Pot1pN–ssDNA complex32. In both cases, these self-recognition interactions seem to stabilize the bent backbone of the ssDNA. The other type of DNA self-recognition (G-T base-pairing) observed in the S. pombe complex is not seen here.

The nucleotide at the very 3′ end, G10, is bound within a pocket formed by two β-strands (β3 and β5) and another por-tion of the DNA. The edge of the base of G10 is surrounded with four hydrogen bond interactions (Fig. 3f) that seem to be account for the highly sequence-specific recognition of G10 (Fig. 1d). The terminal 3′-hydroxyl group of G10 is solvent-exposed, consistent with ability of hPOT1 to accommodate additional 3′ nucleotides in its DNA-binding cleft (albeit with reduced affinity).

Telomeric DNA end-protectionThe crystal structure of the telomeric complex presented here reveals how hPOT1 recognizes its cognate sequence, and provides an explanation for

Figure 2 Structure of the hPOT1V2–ssDNA complex. (a) Experimental electron density map (after density modification) at 1.73-Å resolution showing the stacking interactions of nucleotides G5 and G6 with Phe31, and nucleotide T7 with Tyr161 and Tyr271. The refined model of the protein and ssDNA is superimposed on the map. Contours are drawn at the 1.0-σ level. The DNA is in ball-and-stick representation (carbon, white; nitrogen, blue; oxygen, red; phosphorus, yellow). The protein is green. This panel was generated using BobScript48 and Raster3D49. (b) An electrostatic potential surface representation of hPOT1V2. Positive potential, blue; negative potential, red (at the 10 kT e–1 level). The DNA is in ball-and-stick representation and colored as in a. Electrostatic potentials were calculated with the DNA removed using GRASP50. (c) Ribbon diagram of the hPOT1–ssDNA complex with hPOT1 cyan (β-strands), green (α-helices) and orange (loops). Orange dots, short disordered loop. The DNA is in ball-and-stick representation and colored as in a. The secondary structure elements of the protein are labeled. This panel was generated using MolScript51 and Raster3D49. (d) Schematic representation of the protein-ssDNA stacking interactions. Two OB folds are light blue ellipses. Bases of the ssDNA are yellow bars, phosphodiester groups are purple circles, sugar rings are white pentamers and residues of hPOT1V2 are blue bars; nucleotides of ssDNA, orange labels.

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the binding specificity at an atomic level. To study the hPOT1–ssDNA complex in solution, dimethyl sulfate (DMS) methylation footprinting was used to monitor the accessibility of guanine residues in the complex (Fig. 4a). Oligonucleotide h2T, with an hPOT1-binding site preceded by a nontelomeric leader, provided a G outside the protein-binding region for comparison. The 3′-terminal nucleotide, G10, was strongly protected from methylation and G5 only moderately protected (Fig. 4b). In con-trast, the extent of methylation at the other two positions (G4 and G6) was similar for the hPOT1–h2T complex and the uncomplexed h2T. The control ssDNA methylation patterns showed no difference in the pres-ence or absence of hPOT1, confirming that the methylation protection was DNA sequence-specific.

These DMS methylation footprinting data were consistent with the crystal structure. The base of the strongly protected G10 is buried in a binding pocket and surrounded by four hydrogen bonding interactions (Supplementary Fig. 4 online), and its N7 atom is inaccessible. The base of G5 is sandwiched between the base of G6 and the side chain of Phe31 with its N7 atom partially covered by a hydrogen bonding interaction involving the side chain of Thr48. In contrast, the N7 atoms of G4 and G6 are completely solvent-accessible. Hence, we conclude that the 3′ end of the telomeric DNA is tightly protected by hPOT1 in solution as it is in the crystal structure.

DISCUSSIONModel for chromosome end-cappingThe single-stranded telomeric overhang is the product of telomer-ase, a ribonucleoprotein that synthesizes telomeric DNA by iterative reverse transcription of its own internal RNA template. If the RNA template region is fully copied before telomerase finally dissociates, all

chromosomes should end with the sequence TTAGGGTTAG36,37, which is identical to the specific recognition sequence of hPOT1 (Fig. 5a). Thus, the crystal structure of the hPOT1–hT10 complex provides an atomic-resolution model for chromosome end-cap-ping by hPOT1. It should be noted, however, that chromosome end-capping by POT1

in vivo has thus far been demonstrated only in S. pombe13 and has not yet been tested in human cells.

The structure of the hPOT1–hT10 complex described here also reveals an unanticipated feature: an hPOT1 protein monomer uses two OB folds to bind a decamer telomeric ssDNA (Fig. 5b); this is different from S. pombe Pot1pN, where one OB fold binds six nucleotides. Ten nucleotides are not an integral number of human telomeric repeats. Yet modeling shows that the extra two nucleotides could span the dis-tance to the next repeat, so that long telomeric ssDNA could be coated with every two repeats (12 nucleotides) bound to an hPOT1 monomer (Fig. 5b). Thus, hPOT1 would coat the entire single-stranded overhang and remain in phase with the telomeric sequence.

Comparison with other telomeric proteinsAn unbiased search for structurally homologous proteins using Dali38 revealed that the structure of OB1 is most similar to that of the OB fold of fission yeast (S. pombe) Pot1pN protein32. The two structures can be superimposed with an r.m.s. deviation of 2.0 Å in the positions of 136 out of 140 Cα atoms (Supplementary Fig. 5 online). This is con-sistent with the fact that the conformation of nucleotides T1–G4 is also

Figure 3 Protein-ssDNA and ssDNA-ssDNA interactions in the hPOT1V2–ssDNA complex. (a–f) Detailed interactions between each nucleotide and its interacting protein residues.Both hPOT1V2 and the DNA are in ball-and-stick representation. hPOT1V2 is colored with carbon cyan, nitrogen blue and oxygen red. The ssDNA is colored as in Figure 2a. Protein-DNA intermolecular hydrogen bonds are dotted yellow lines, and ssDNA intramolecular hydrogen bonds are dotted green lines. All diagrams were generated using MolScript51 and Raster 3D49.

Figure 4 Telomeric DNA end-binding by hPOT1. (a) Schematic representation of the methylation footprint patterns generated by hPOT1. The nucleotide numbering of h2T and the control oligonucleotide is at the top of the DNA sequences. Squares with white and gray shading represent moderate and extensive protection from methylation, respectively. (b) The 5′-labeled h2T and control oligonucleotide were incubated with or without hPOT1V1, and subjected to DMS methylation and pyrrolidine cleavage. The methylation footprint patterns were analyzed by urea-PAGE. Gc, a G residue in the 5′ nontelomeric sequence serves as an internal control for loading differences. The black ellipse denotes the heavy protection of G10. The experiment was repeated with hPOT1V2 with equivalent results (data not shown).

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most similar to that of T3–C6 in the crystal structure of the S. pombe Pot1pN–ssDNA complex. Although OB2 is structurally more divergent than OB1, its closest structural homolog can be detected by Dali and is one of the RPA OB folds (DBD-B)30, with an r.m.s. deviation of 2.4 Å for 105 Cα atoms.

Efforts to prepare the OB1 fragment of hPOT1 (residues 1–150) yielded insoluble protein (data not shown), suggesting that unlike S. pombe Pot1pN32, hPOT1 OB1 on its own may be largely unfolded and may require an interface with the second OB fold for stabil-ity. The fact that OB1 of S. pombe Pot1 binds its telomeric single-stranded DNA with a Kd of 83 nM suggests that even if S. pombe Pot1 has a second OB fold, it is not required for high-affinity binding as in hPOT1.

hPOT1 OB1-OB2 packing involves both hydrophobic van der Waals contacts and hydrogen bond networks, and buries a total surface area of 1,000 Å2 (Supplementary Fig. 5 online). The residues that form the interface are highly conserved across vertebrate species (Supplementary Fig. 6 online). Given that all vertebrates share the same telomeric repeat sequence, TTAGGG, these observations indicate that the prearrange-ment of the two OB folds is important for Pot1 protein functions in all vertebrates.

UP1 (hnRNP A1), best known as an RNA-binding protein, can also bind single-stranded telomeric DNA. The crystal structure of the UP1 telomeric single-stranded DNA complex39 shows that DNA bases inter-act with two RNA recognition motifs (RRMs) in a way superficially similar to that of the hPOT1–ssDNA structure presented here. On the other hand, the self-recognition of DNA and 3′ end-protection observed here are not present in UP1.

Telomerase regulationFor a chromosome end to be extended by telomerase, the 3′ end of the DNA must be accessible to pair with the alignment region pre-ceding the RNA template40. Examination of the hPOT1–DNA crystal structure shows that the 3′-terminal guanine base is buried in the protein, which should render it inaccessible to telomerase. Indeed, in vitro telomerase assays confirm that equimolar hPOT1 prevents an oligonucleotide ending in GGTTAG from serving as a substrate (A. Zaug , M.L., E.P. and T.R.C., unpublished data). Thus, hPOT1 has the intrinsic ability to inhibit the action of telomerase, as predicted previously23. The mechanism by which the chromosome tail is handed off from hPOT1 to telomerase would then involve components beyond those present in a simple in vitro assay, providing an important area for future study.

Possible higher order structuresIt has been proposed that at least a portion of hPOT1 is in a complex with TRF1 and PTOP/PIP1 (refs. 23,41,42). The interaction with PTOP/PIP1 is mediated by hPOT1’s C-terminal region41,42, and we have shown here that this domain has no effect on hPOT1’s ssDNA-binding activity. This supports the conclusion that hPOT1 could cap the very end of the chromosome in the manner seen in the crystal structure, even if it is part of a higher-order complex.

Our crystallographic and biochemical data show that hPOT1 by itself can cap telomere 3′ ends. However, hPOT1 may contribute to telomere function in other ways in vivo. For example, EM has revealed that the entire human telomere (5–10 kilobases of double-stranded DNA) can form a large loop called a T loop, with the single-stranded terminus paired with an internal region43,44. Formation of a T loop displaces an internal segment of single-stranded TTAGGG repeats (D loop), which could bind hPOT1. Thus, hPOT1 could carry out some of its functions in concert with T loops in vivo.

METHODSProtein expression and purification. The genes for hPOT1V1 and hPOT1V2 were cloned into a modified Bac-to-Bac vector containing an N-termi-nal glutathione S-transferase (GST) tag preceding the multiple cloning sites (Gibco). For protein expression, Sf9 cells were infected at ∼1.5 × 106 cells ml–1 with a multiplicity of infection of 1 plaque-forming unit ml–1 recombinant baculovirus. The cells were harvested after 48 h by centrifugation and stored at –80 °C. Approximately 20 g of cells (wet weight) were resuspended in 50 ml of lysis buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM 2-mercaptoethanol, 5 mM benzamidine, and 1 mM PMSF) and incubated on ice for 15 min. The cells were lysed by sonication and cell debris was removed by centrifugation. The supernatant was applied to glutathione Sepharose-4b beads and eluted with 15 mM glutathione. Proteins were further purified by gel filtration chromatography (Superdex 200). For proteins used in the biochemical studies, 3C protease was then added to remove the N-terminal GST tag followed by gel filtration chromatogra-phy (Superdex 200). Proteins at this stage were >99% pure and were concentrated to 1 mg ml–1 by Centricon 10 (Millipore) and stored at –80 °C. Alternatively, for materials used in crystallization, gel filtration–purified GST-hPOT1V2 was mixed with ssDNA (TTAGGGTTAG) before addition of 3C protease. Then the hPOT1V2–ssDNA complex was subjected to a final gel filtration chromatography step (Superdex 200) and was concentrated to 10 mg ml–1 by Centricon 10 and stored at –80 °C.

Gel mobility shift assay. hPOT1V1 or hPOT1V2 in binding buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5% (v/v) glycerol, and 1 mM DTT) was incubated with 5′-32P-labeled telomeric ssDNA at room temperature for 60 min. Then the mixtures were directly loaded onto a nondenaturing 4–20% (w/v) polyacrylamide gel as described22.

Crystallization, data collection and structure determination. Crystals were grown by hanging-drop vapor diffusion at 16 °C using a protein/ssDNA molar ratio of 1:2 by adding 1 molar ratio of ssDNA to the hPOT1V2–ssDNA complex. The precipitant (well solution) contained 100 mM trisodium citrate, pH 5.4–5.8, 0.6 M ammonium sulfate and 10 mM DTT. The crystals belong to the ortho-rhombic space group C2221 with unit cell dimensions of a = 102.0, b = 103.2 and c = 71.66 Å. The asymmetric unit contains a single hPOT1V2–ssDNA complex.

Figure 5 Model showing how hPOT1 could coat the entire 3′-end single-stranded overhang of a chromosome and inhibit telomerase activity. (a) Telomerase synthesizes and hPOT1 binds repeats of GGTTAG, not TTAGGG. The template of the human telomerase RNA component52 is reverse-transcribed to give the specific ssDNA sequence recognized by hPOT1. (b) Schematic illustration showing how hPOT1 might coat the entire 3′ single-stranded overhang. Blue and yellow ovals, the two OB folds of a single hPOT1 monomer. Kinked red bar, telomeric ssDNA. Each hPOT1 molecule binds two telomeric repeats. The 3′ end of the overhang is prevented from interacting with telomerase. C-terminal domain of hPOT1 (not shown in the crystal structure), green oval.

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Crystals were gradually transferred into a harvesting solution containing 100 mM sodium citrate, pH 5.4, 1 M ammonium sulfate, and 4.3 M sodium formate, then flash-frozen in liquid nitrogen for storage and data collection under cryogenic conditions (100 K). Data were collected at the Advanced Light Source Howard Hughes Medical Institute beamlines 8.2.1 and 8.2.2 using an ADSC Quantum Q4 detector and processed using HKL2000 (ref. 45). MAD data from the mercury and bromine derivatives were used to obtain initial phases. Three mercury atoms and three bromine atoms were located by anomalous Patterson and difference Fourier methods, using CNS46. These positions were refined and the MAD phases of each derivative calculated using CNS. However, neither mercury nor bromine MAD phases could generate an electron density map good enough for model building. The phase combination of those two MAD data sets followed by solvent flattening allowed a model to be automatically built into the modified experimental electron density using ARP/wARP47; the model was then refined using simulated-annealing and posi-tional refinement (CNS46), with manual rebuilding. The final model includes 291 amino acids, 10 nucleotides and 255 water molecules.

DMS methylation footprinting. 5′-labeled h2T or control oligonucleotide was incubated with hPOT1V1 or hPOT1V2 in 30 µl of 50 mM Tris, pH 8.0, 15 mM NaCl, and 1 mM DTT for 60 min at room temperature. Then 3 µl of 10% (v/v) DMS was added and incubated for 15 min at room temperature. The methyla-tion reaction was stopped by the addition of 6 µl of stop buffer (1 µg µl–1 yeast tRNA, 0.1 mM EDTA, 2.5 M 2-mercaptoethanol, and 0.5% (w/v) SDS in TE, pH 7.5). The reaction mixture then was phenol-chloroform extracted, followed by ethanol precipitation. The pellet containing the methylated DNA was resus-pended in 25 µl of distilled water, and then cleaved in 50 µl of 1 M pyrrolidine

for 30 min at 90 °C and dried in the speed vacuum (Savant). The cleaved DNA was washed twice with distilled water, dissolved in 25 µl of 93% (v/v) formamide and assayed by 8% (w/v) polyacrylamide, 7-M urea gel electrophoresis.

Coordinates. The atomic coordinates and structure factors of hPOT1V2–ssDNA complex have been deposited in the Protein Data Bank (accession code 1XJV).

Note: Supplementary information is available on the Nature Structural & Molecular Biology website.

ACKNOWLEDGMENTSWe thank L. Chen and D. Theobald (University of Colorado-Boulder) for insightful discussions and C. Ralston and G. McDermett (Advanced Light Source) for help in data collection. We thank K. Christensen and the tissue culture core facility of the University of Colorado Cancer Center for virus amplification and protein expression. This work was supported in part by a grant from the US National Institutes of Health. M.L. is an Agouron/Paul Sigler research fellow of the Helen Hay Whitney Foundation.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 30 July; accepted 25 October 2004Published online at http://www.nature.com/nsmb/

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Table 1 Data collection, phase determination and refinement statistics

Hgλ1 Hgλ2 Hgλ3 Hgλ4 Brλ1 Brλ2 Brλ3 (remote 1) (inflection) (peak) (remote 2) (remote) (inflection) (peak) Native

Data collection

Wavelength (Å) 1.0255 1.0094 1.0088 0.9927 0.9067 0.9206 0.9202 1.1070

Resolution (Å) 42–2.4 42–2.4 42–2.4 42–2.4 42–2.3 42–2.3 42–2.3 1.73

Observations 206,137 208,897 194,881 211,201 240,068 247,512 241,596 271,324

Unique reflections 15121 15,058 15,029 15,270 17,437 17,021 17,421 38,238

Rsym (%) 8.8 9.2 8.8 9.7 6.6 6.4 6.3 4.1

Average I / σ 10.6 11.3 11.9 10.9 13.2 13.8 13.8 15.2

Completeness (%) 99.7 99.6 99.3 99.3 99.9 99.9 99.9 95.4

Redundancy 13.6 13.9 13.0 13.8 13.8 14.5 13.9 7.1

MAD analysis

No. of sites 3 3 3 3 3 3 3

Phasing power (dispersive) N/A 0.5 0.52 0.48 N/A 0.24 0.31

Rcullis (dispersive) N/A 0.86 0.87 0.87 N/A 0.94 0.96

FOM (dispersive) N/A 0.12 0.12 0.11 N/A 0.04 0.07

Phasing power (anomalous) 0.33 0.32 0.34 0.23 0.21 0.32 0.07

Rcullis (anomalous) 0.88 0.91 0.91 0.92 0.94 0.91 0.98

FOM (anomalous) 0.07 0.09 0.09 0.06 0.04 0.05 0.02

Hg

Br

Hg-Br phase

combinationResolution (Å), Final 42–2.4 42–2.3 50–2.4 (50–1.73)a FOM, Final 0.30 0.13 0.41 (0.76)a

Refinement

Rcryst (%) 22.8

Rfree (%) 23.7

R.m.s. deviations

Bond length (Å) 0.006

Bond angle (°) 1.39

B-factor (Å2) 1.56

N/A, not applicable. Rfree is calculated for a randomly chosen 5% of reflections; Rcryst is calculated for the remaining 95% of reflections used for structure refinement. aNumbers in parentheses represent the values of resolution and FOM after phase extension and density modification (solvent flattening).

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