How telomeric POT1 avoids RNA to achieve specificity for single-stranded DNA

Jayakrishnan Nandakumar, Elaine R. Podell, and Thomas R. Cech1

Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215

Edited by Titia de Lange, The Rockefeller University, New York, NY, and approved November 9, 2009 (received for review September 25, 2009)

The POT1-TPP1 heterodimer, the major -specific single- sites consensus sequence ðPyÞnNPyAG∕G (Py ¼ pyrimidine, n is stranded DNA-binding protein in mammalian cells, protects chro- an integer >1, and “∕” indicates the splice site) is essentially mosome ends and contributes to the regulation of . The satisfied by the telomeric repeat sequence GTTAGG. Because 30 recent discovery of telomeric RNA raises the question of how POT1 splice sites are present on most pre-RNAs, their concentration in faithfully binds telomeric ssDNA and avoids illicit RNA binding that the nucleus is expected to exceed that of 30 ss G-rich DNA tails. could result in its depletion from . Here we show through In addition to TERRA, the number of RNA r(UUAGGGU binding studies that a single deoxythymidine in a telomeric repeat UNG) sequences that match the POT1-binding site expected to dictates the DNA versus RNA discrimination by human POT1 and occur randomly in the nucleus of a mammalian cell is ∼1; 300– mouse POT1A. We solve the crystal structure of hPOT1 bound to 20; 000 (25), which is greater than or equal to the number of d DNA with a ribouridine in lieu of the critical deoxythymidine (TTAGGGTTAG) binding sites of hPOT1 (∼1; 500) in a human 0 and show that this substitution results in burying the 2 -hydroxyl cell (2, 4) (calculation in Supporting Information). Despite these group in a hydrophobic region (Phe62) of POT1 in addition to potential RNA-binding alternatives, POT1 faithfully and strongly eliminating favorable hydrogen-bonding interactions at the POT1– binds telomeric DNA showing no apparent affinity for any of nucleic acid interface. At amino acid 62, Phe discriminates against these RNAs (6, 14, 23, 26). We wondered how mammalian POT1 RNA binding and Tyr allows RNA binding. We further show that displays such robust intolerance to RNA. Here, through binding TPP1 greatly augments POT1’s discrimination against RNA. experiments with mixed DNA-RNA oligonucleotides and a high- resolution crystal structure, we show that a single ribouridine in crystal structure ∣ DNA–protein interaction ∣ POT1-TPP1 ∣ telomere lieu of a deoxythymidine at the fourth position (rU4 instead of dT4) of a telomeric repeat sequence d(GGTTAGGGTTAG) is elomeres are protein-DNA complexes that comprise the the primary determinant of RNA discrimination by hPOT1. Ttermini of linear and help maintain the integrity We further show that POT1-TPP1 displays remarkably greater of eukaryotic genomes (1). Telomeric DNA typically consists of a intolerance towards RNA than POT1 alone, uncovering a pre- large number of short repeats of dsDNA ending with a single- viously undescribed function for the TPP1 protein. stranded (ss) G-rich 30 overhang (2–4). Specialized telomeric bind to the ds and ss regions of telomeric DNA to pre- Results vent inappropriate degradation and fusion events at dT4 Dictates POT1 Specificity for DNA. We observed that mouse ends (5). One such protein, protection of telomeres 1 (POT1), 0 POT1A, the essential POT1 paralog in mice (7), serves as an ex- binds specifically to the ss G-rich 3 tail of chromosomes (6–11). cellent source of recombinant mammalian POT1, because it can Human POT1 (hPOT1) and hPOT1V2 (a splice variant of be obtained readily from overexpression in bacteria (unlike hPOT1 composed of its DNA-binding domain, used extensively hPOT1, which is isolated after expression in baculovirus-infected to characterize POT1 structurally and biochemically) bind telo- insect cells). To examine the effects of ribonucleotide substitution meric DNA with high affinity (KD ∼ 10 nM) and base specificity

within the telomeric sequence on mammalian POT1 binding, we BIOCHEMISTRY (10). POT1 is conserved among all mammals and has functional carried out a parallel binding analysis of hPOT1 and mPOT1A Oxytricha nova Tetrahy- homologs in other species such as (12), with oligonucleotides that contain two telomeric repeats termi- mena thermophila Schizosaccharomyces pombe (13), and (6, 14). nating in TAG-30, which is optimal for mPOT1A and hPOT1 Arabidopsis thaliana A sequence-related protein in the plant binding (27, 28). associates with the telomerase ribonucleoprotein rather than – Like hPOT1 (6), mPOT1A failed to bind an all-RNA telomeric the telomeric DNA (15 17). dodecamer R12 under conditions in which it formed a stable TPP1, another telomeric protein, binds POT1 and is critical for A – complex with D12 (Fig. 1 ; oligonucleotide sequences in Table1). POT1 recruitment to telomeres (18 21). Although human TPP1 To probe whether the DNA versus RNA specificity of POT1 is (hTPP1) does not bind telomeric DNA directly, it increases the derived from determinants found throughout the ssDNA or from affinity of hPOT1 for telomeric ssDNA (21). Additionally, the certain “hot spots” in the dodecameric sequence, we measured hPOT1-hTPP1 complex increases the processivity of telomerase, the affinity of hPOT1 and mPOT1A for variants of D12 that have the unique reverse transcriptase that maintains telomere length 0 stretches of three consecutive ribonucleotides in the telomeric by catalyzing the synthesis of telomeric DNA at 3 ends of chro- sequence. Both hPOT1 and mPOT1A formed a discrete complex mosomes (21). hTPP1-N, an N-terminal fragment of hTPP1 thatincludesanOligonucleotide/oligosaccharide binding(OB)do- main and the POT1-binding domain, fully recapitulates hTPP1’s Author contributions: J.N. designed research; J.N. and E.R.P. performed research; J.N. POT1-ssDNA-binding-stimulation and telomerase processivity- and T.R.C. analyzed data; and J.N. and T.R.C. wrote the paper. enhancement functions (21). The authors declare no conflict of interest. TERRA is noncoding RNA-containing multiple G-rich This article is a PNAS Direct Submission. telomeric repeats transcribed from chromosome ends (22–24). Freely available online through the PNAS open access option. TERRA is found in mammals and budding yeast and implicated Data deposition: The atomic coordinates and structure factors have been deposited in the in the regulation of telomerase and in chromatin remodeling. , www.pdb.org (PDB ID codes 3KJO and 3KJP). TERRA in humans is 100 bases to 9 kilobases long (22, 24). 1To whom correspondence should be addressed. Email: [email protected]. 30 In contrast, the ss G tails on the ends of human chromosomes This article contains supporting information online at www.pnas.org/cgi/content/full/ 0 are only 130–275 nucleotides long (2, 4). The pre-mRNA 3 splice 0911099107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0911099107 PNAS ∣ January 12, 2010 ∣ vol. 107 ∣ no. 2 ∣ 651–656 Downloaded by guest on September 23, 2021 Fig. 1. Effect of ribonucleotide substitution on POT1-ssDNA binding. EMSA of mPOT1A (A) and hPOT1 (B) with 32P-labeled dodecameric ssDNA-RNA mixed oligonucleotides of telomeric sequence. Binding mixtures contained 1 nM 32P-labeled oligonucleotides and 1.5 nM mPOT1A (A)or20nM32P-labeled oligonucleotides and 40 nM hPOT1 (B). The sequences of the oligonucleotides are detailed in Table 1. (C) Filter-binding experiments of mPOT1A with 32P-labeled ssDNA-RNA of the indicated sequence (ribonucleotides are depicted in red) were done in duplicate, and the mean of the fraction of mPOT1A-bound ssDNA-RNA was plotted against mPOT1A concentration. Error bars represent the standard deviation of the two measurements.

with 1–3R, 7–9R, and 10–12R but not with 4–6R (Fig. 1A and B). the complex formed by that dodecamer to the KD for the analo- To evaluate whether the decreased binding seen with 4–6R arose gous complex formed by D12 (designated as “fold increase of from the presence of one particular ribonucleotide, we tested KD” in Table 1). Consistent with our gel-shift analysis (Fig. 1A D12 variants with a single ribonucleotide at either the fourth and B), 10–12R did not show a binding defect when compared (4R), fifth (5R), or sixth (6R) position from the 50 end. Fig. 1A to D12 (Fig. 1C and Table 1). hPOT1-1–3R and hPOT1-7–9R and B show that with hPOT1 and mPOT1A, 5R and 6R had had 2-fold and 5-fold increases of KD, respectively. In agreement binding profiles similar to that shown by D12. In contrast, the with the gel-shift analysis, the most significant binding defect was POT1-4R complex dissociated during gel electrophoresis much seen in the case of 4–6R (Table 1). With hPOT1, 4–6R binding like POT1-4–6R. Hence, a single ribonucleotide (rU instead of was decreased by 15-fold. The deleterious effect of ribonucleo- dT) at position 4 interferes with POT1 binding. tide substitutions in positions 4–6 was further magnified in the case of mPOT1A, where 47-fold reduced binding was observed. Quantitation of the Deleterious Effects of Ribonucleotide Substitution Single ribonucleotide substitutions in the context of D12 at on POT1-ssDNA Binding. Using a filter-binding assay, we deter- positions 4, 5, and 6 (4R, 5R, and 6R) were assessed to quantify mined the KD of hPOT1-D12 to be 6.3 3.5 nM and that for the contributions of each of these positions to DNA specificity. mPOT1A-D12 to be 0.9 0.3 nM, values that agree well with Again, in agreement with the binding data shown in Fig. 1A and previous reports (10, 28). To quantify the defect in binding for B, the greatest defect in binding is seen for 4R (4-fold for hPOT1 a particular dodecamer, we calculated the ratio of the KD for and 22-fold for mPOT1A; Table 1). Because rU differs from dT

Table 1. KD’s for hPOT1, hPOT1-hTPP1-N, mPOT1A, and mPOT1A-mTPP1-N with DNA-RNA mixed dodecameric oligonucleotides 1TOPh OPh T1 + N-1PPTh A1TOPm Pm OT1A + Tm PP1N

Stimulation Stimulation Name of Fold Fold of binding Fold Fold of binding

oligo- Nucleic acid KD (nM) Increase KD (nM) Increase by KD (nM) Increase KD (nM) Increase by f f s f f s nucleotide sequence of KD of KD hTPP1-N of KD of KD mTPP1N

D12 GGTTAGGGTTAG 6.3 ± 3.5 (1) 0.1 ± 0.02 (1) 63 0.9 ± 0.3 (1) 0.04 ± 0.01 (1) 23 1-3R GGUTAGGGTTAG 15.1 ± 2.1 2 1.9 ± 0.9 19 8 3.2 ± 0.2 4 0.8 ± 0.6 20 4 4-6R GGTUAGGGTTAG 92.4 ± 17 15 34.9 ± 2.1 349 3 42.5 ± 15.4 47 94 ± 23.6 2350 0.5 7-9R GGTTAGGGUTAG 31.7 ± 10.5 5 4.9 ± 1.6 49 6 2.5 ± 0.1 3 1.4 ± 1.2 35 2 10-12R GGTTAGGGTUAG 5 ± 2.1 1 0.8 ± 0.14 8 8 0.7 ± 0.2 1 0.1 ± 0.02 3 7 4R GGTUAGGGTTAG 24.3 ± 2.5 4 11.9 ± 2.1 119 2 19.4 ± 3.3 22 18.7 ± 0.4 468 1 5R GGTTAGGGTTAG 6.9 ± 3.8 1 1.4 ± 0.1 14 4 2.3 ± 1.3 3 n.d - - 6R GGTTAGGGTTAG 2.6 ± 1.9 0.4 0.5 ± 0.1 5 5 0.6 ± 0.3 1 n.d - - 4dU GGT(dU)AGGGTTAG 8.9 ± 0.4 1 0.7 ± 0.1 7 12 1.7 ± 0.4 2 1.2 ± 0.1 30 1 4rT GGT(rT)AGGGTTAG 9.6 ± 1.7 2 1.4 ± 0.3 14 7 2.7 ± 0.4 3 2.5 ± 0.4 63 1 R12 GGUUACGGUUAC ~1200a ~190 ~2250a ~31250 ~0.5 ~300a ~333 ~900a ~22500 ~0.3 4D GGUTACGGUUAC ~300a ~48 ~350a ~4860 ~1 60.2 ± 24 67 ~110a ~2750 ~0.5 4-5D GGUTACGGUUAC ~110a ~18 ~190a ~2600 ~2 16.4 ± 0.6 18 ~100a ~2500 ~0.2

Ribonucleotides are underlined and shown in red. KD values represent averages of two independent experiments ± the standard error of the two experiments.

n.d: KD was not determined

f Ratio of KD with the oligonucleotide in question and the KD with D12

s Ratio of KD with POT1 and KD with POT1-TPP1-N

a Because incomplete binding was observed at 100 nM protein, the KD is only approximate

652 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0911099107 Nandakumar et al. Downloaded by guest on September 23, 2021 by having a 20-OH and by lacking a 5-methyl group on the base, and Material and Methods). Structural comparison showed that we tested oligonucleotides containing an rTand a dU at position the protein backbone and side chains of 1XJV and hPOT1V2- 4. Compared to D12, both 4rT and 4dU showed modest binding D12 (rmsd ¼ 0.2 Å over 291 Cα atoms) superimpose well, as do defects with mPOT1A (4rT showing a 3-fold defect and 4dU the 10 nucleotides common to the two structures (Fig. S1A). The showing a 2-fold defect). With hPOT1, no significant binding eleventh nucleotide (dG2) in our hPOT1V2-D12 structure stacks defect was observed with 4rTor 4dU. We conclude that the effects against dT3 and makes no other contacts to the DNA or protein of the 5-methyl and 20-H groups on dT4 are not additive in terms (Fig. S1B). No electron density was observed for the twelfth of binding energy (or not multiplicative in terms of KD) but rather nucleotide, dG1. We conclude that D12 and d(TTAGGGTTAG) synergistic in giving rise to DNA specificity for POT1. have similar modes of binding to POT1, and hence ribonucleotide hPOT1 binds the all-RNA oligonucleotide R12 with substitutions at the 10 conserved positions of D12 and d(TTAGG KD ∼ 1; 200 nM, whereas it binds D12 with KD ¼ 6.3 nM. Given GTTAG) are expected to have similar effects on POT1 binding. that dT4 makes the largest single contribution to DNA specificity Indeed, hPOT1 binds d(TTAGGGTTAG) with 10-fold greater for hPOT1, we asked whether introducing dT4 in the context of affinity than it binds dTrUd(AGGGTTAG) (Fig. S2). R12 (4D, Table 1) could rescue POT1 binding. Indeed, 4D bound To analyze the structural basis for the binding defect seen in POT1 with 4 times greater affinity than R12 (KD ∼ 300 nM). It oligonucleotides containing the 4rU substitution, we crystallized seemed possible that reinforcing the DNA-like 20-endo sugar puck- the hPOT1V2-dTrUd(AGGGTTAG) complex and solved the er of dT4 in 4D by introducing an adjacent deoxyribonucleotide structure at 1.8 Å resolution (Supporting Table 1). The final re- ∕ ¼ 0 228∕0 238 2F − F could lead to further POT1-binding recovery. This was the case, be- fined model had an Rcrystal Rfree . . .A o c map cause hPOT1 bound 4–5D (Table 1) with a KD of ∼110 nM. How- (Fig. 2A) andasimulated annealing compositeomitmap (Fig. S3A) ever, binding remained ∼17-fold weaker than that of hPOT1-D12, generated using the CNS program (29) unambiguously confirmed providing additional evidence that the difference between RNA the position of the O20 of rU4 (Fig. 2A). There were no significant and DNA binding is not completely localized to a single position. changes to the protein backbone (rmsd ¼ 0.2 Å over 289 Cα atoms), although subtle changes to the nucleic acid backbone were Structural Basis for DNA versus RNA Specificity of POT1. The binding observed when compared to 1XJV (Fig. 2D, E, and F). The poten- studies reported here use oligonucleotides that include two 50 G tial role of nucleic acid backbone geometry in RNA discrimination nucleotides not present in the hPOT1V2-d(TTAGGGTTAG) by POT1 and by POT1-TPP1 is detailed in Discussion. structure [Protein Data Bank (PDB): 1XJV] (10). To ask whether The O20 of rU4 in the hPOT1V2-dTrUd(AGGGTTAG) struc- the presence of the additional G nucleotides alters the POT1- ture (Fig. 2B) is located at a distance of 3.3 Å from the ε-C of the ssDNA interface, we crystallized hPOT1V2 in complex with aromatic side chain of Phe62, which is involved in a π-sandwich D12 and solved the structure at 1.8 Å (See Supporting Table 1 with the bases of rU4 and dT3. We suggest that positioning of the BIOCHEMISTRY

2F − F 1σ Fig. 2. Structural basis for RNA discrimination by hPOT1. (A) A stereo view showing the electron density ( o c) around rU4 contoured at obtained from rigid body refinement of hPOT1V2-d(TTAGGGTTAG) (PDB: 1XJV) against crystallographic data of hPOT1V2-dTrUd(AGGGTTAG) at 1.8 Å. The final refined model is superimposed on the map as a stick model in Corey–Pauling–Koltun (CPK) atomic coloring. Density for the O20 is clearly defined in the electron density. The polar and stacking interactions involving dT3, rU4/dT4, dA5, and dG6 in the hPOT1V2-dTrUd(AGGGTTAG) (B) and the hPOT1V2-d(TTAGGGTTAG) (PDB: 1XJV) (C) structures are shown. The two-headed red arrow in (B) indicates a van der Waals contact between the O20 of rU4 and the Phe62 of hPOT1. (D–F) hPOT1V2-dTrUd(AGGGTTAG) (red) and hPOT1V2-d(TTAGGGTTAG) (green) structures were superposed to highlight differences in the nucleic acid backbone along nucleotides dT3-dG6 (D), dG7-dT9 (E), and dT10-dG12 (F). The double-headed black arrows indicate instances of significant DNA-C20–protein proximity (<4.5 Å). Note that the numbering scheme for the nucleic acid in this paper (and associated PDBs) is G(1)G(2)T(3)T(4)A(5)G(6)G(7)G(8)T(9)T(10)A(11) G(12). We have numbered the DNA strand for hPOT1V2-d(TTAGGGTTAG) according to this scheme, although previous references, including PDB: 1XJV, adopt the following numbering scheme: [T(1)T(2)A(3)G(4)G(5)G(6)T(7)T(8)A(9)G(10)].

Nandakumar et al. PNAS ∣ January 12, 2010 ∣ vol. 107 ∣ no. 2 ∣ 653 Downloaded by guest on September 23, 2021 O20 in close proximity to Phe62, which is part of a hydrophobic side chains of POT1 (Fig. 2D, E, and F), are in predominantly patch of hPOT1 (Fig. 3A), precludes solvation of the 20OH group. hydrophilic environments (Fig. 3B, C, and D). The fourth, rU4 that The conformation adopted by the rU4 ribose sugar, which is very stacks against Phe62, is in a hydrophobic environment (Fig. 3A), as different from that of the dT4 sugar, perturbs the conformation of discussed above. It is possible that ribonucleotide substitutions at the phosphate backbone link to dT3 (compare Fig. 2B and C, and positions dG6, dT9, and dG12 of a telomeric oligonucleotide do see superposition in Fig. 2D). This perturbation results in the re- not adversely affect hPOT1 binding because the polar O20 (cova- arrangement of the base of dT3 in the rU4-containing complex, lently bonded to C20) is well accommodated in the hydrophilic such that the hydrogen bond between N3 of dT3 and the γ-O of environment provided by hPOT1 (Fig. 3B, C, and D). In contrast, Thr41 (seen in 1XJV) is lost and the Thr41 side chain adopts an the dT4 to rU4 change that introduces an O20 and removes a 5- alternative rotameric conformation. Additionally, a possible methyl group (both changes reduce hydrophobicity) places rU4 hydrogen-bonding interaction between the carboxylate of Asp42 in a predominantly hydrophobic environment (Fig. 3A), possibly and the O4 of dT3 in 1XJV (requiring protonation of either the contributing to the energetic destabilization reflected in the bind- δ-O of Asp42 or the O4 of dT3) is no longer observed in the rU4- ing defect of hPOT1 with 4R and 4–6R (Fig. 1 and Table 1). containing complex. In addition to the lost direct protein–nucleic We hypothesized that, in the context of the POT1 structure, the acid interactions, two water-mediated interactions that are pres- hydrophobic environment created by phenylalanine discriminates ent in 1XJV are absent in the hPOT1V2-dTrUd(AGGGTTAG) against ribonucleotide binding, whereas the more hydrophilic structure (Fig. S3B and C). environment created by tyrosine allows ribonucleotide binding. To With the exception of Thr41, no significant differences in the test this hypothesis, we expressed and purified mPOT1A-F62Y, protein side-chain conformations between 1XJV and the rU4- mPOT1A-Y89F, mPOT1A-Y161F, and mPOT1A-Y223F mu- containing structure are seen at the protein–nucleic acid inter- tants and compared their binding affinities for ribonucleotide- face. Superposition of the nucleotides from the two structures containing oligonucleotides versus D12 (Fig. 3E–H). Interestingly, shows that all bases involved in base stacking (rU/dT4, dG6, the F62Y mutant showed only a 2-fold binding defect with 4–6R dG7, dT9, dG12) with aromatic POT1 side chains overlay pre- compared to the 47-fold defect seen with wild-type protein (Fig. 3E cisely on each other with the exception of rU4/dT4 (Fig. 2D, and Table1). In further support of our hypothesis, Y89FandY161F E, and F). For nucleotides not involved in base stacking directly showed modest increases in binding defects with 4–6R (70-fold with aromatic amino acids, the superposition is less perfect. versus 47-fold for wild-type; Fig. 3F) and 7–9R, respectively (7-fold To gain insight as to why POT1 is specifically sensitive to RNA versus 3-fold for wild-type; Fig. 3G and Table 1). Although Y223F substitution at position 4 and not at other positions in the se- behaved like a wild-type protein, showing no binding defect with quence, we inspected the vicinity of the C20 (the atom that bears 10–12R (Fig. 3H), it is possible that further increase in hydro- the 20OH of RNA) for each nucleotide in the hPOT1V2-dTrUd phobicity around Tyr223 is required to observe an increase in (AGGGTTAG) structure (Fig. 3). Four nucleotides (rU4, dG6, RNA discrimination. dT9, and dG12) involved in base stacking with POT1 have their 0 C2 atoms within 4.5 Å of their cognate aromatic protein side TPP1 Binding Greatly Enhances Discrimination against RNA by POT1. chains (Fig. 2D, E, and F). We observe that three of these (dG6, TPP1 is the in vivo protein-binding partner of POT1. In vitro dT9, and dG12), which have their bases stacking against tyrosine binding experiments have shown that, although TPP1 by itself

Fig. 3. Hydrophobicity of Phe62 is critical for RNA discrimination. The crystal structure of hPOT1V2-dTrUd(AGGGTTAG) is shown as a surface representation of the protein and a stick representation of rU4 (A), dG6 (B), dT9 (C), or dG12 (D). The protein is colored according to hydrophobicity of the amino acids [according to the Kyte–Doolittle scale (36)] such that blue to white to dark orange represents increase in hydrophobicity. For clarity, only the pertinent nucleotide (in CPK coloring convention) of the nucleic acid of hPOT1V2-dTrUd(AGGGTTAG) is shown in each panel. The appropriate O20,C20, and aromatic stacking residues of hPOT1 are indicated. (E–H) Binding data and curve fits for mPOT1A-F62Y with D12 and 4–6R (E), mPOT1A-Y89F with D12 and 4–6R (F), mPOT1A-Y161F with D12 and 7–9R (G), and mPOT1A-Y223F with D12 and 10–12R (H). Binding curves of D12 with mPOT1A mutants are shown as solid blue lines and those with wild-type mPOT1A as dotted blue lines. Binding curves of indicated ribonucleotide-substituted oligonucleotides with mPOT1A mutants are shown as solid red lines and those with wild-type mPOT1A as dotted red lines. Error bars represent the standard deviation of two independent measurements. The ratio of the KD with a particular oligonucleotide and KD with D12 for mPOT1A mutants is indicated in black and that for wild-type is indicated in gray.

654 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0911099107 Nandakumar et al. Downloaded by guest on September 23, 2021 does not bind ssDNA, the POT1-TPP1 complex exhibits an ∼10- Inspection of the “fold increase of KD ” columns in Table 1 reveals fold greater affinity for telomeric DNA than POT1 alone (21). that discrimination against a particular RNA-containing oligonu- We performed filter-binding experiments of hPOT1 and mPO- cleotide by hPOT1 (and mPOT1A) is significantly larger in the T1A with the various dodecamers listed in Table 1 in presence of hTPP1-N (and mTPP1-N) than in its absence. Most the presence of hTPP1-N (21) and mTPP1-N, respectively (see notably, hPOT1-4R shows only a 4-fold increase of KD due to Materials and Methods). As expected, the addition of TPP1-N in- ribo-substitution, whereas hPOT1-TPP1-N-4R shows an increase creases the affinity of POT1 for D12 (compare KD for D12 with in KD of 119-fold. These data imply that the discrimination hPOT1 and with hPOT1 þ TPP1-N in Table 1 and similarly for against RNA of telomeric sequence is much greater for POT1 D12 with mPOT1A and with mPOT1A þ mTPP1-N; also see when it is bound to TPP1 and that the mechanism of RNA Fig. 4A). Surprisingly, substitution with a single ribonucleotide discrimination contributed by TPP1 binding is distinct from that (rU4) alters the TPP1 dependence profile entirely. The binding exhibited by POT1 alone. curves for mPOT1A-4R in the presence and absence of mTPP1-N are virtually superimposable as are the curves for the correspond- Discussion ing human proteins (Fig. 4B and C). Hence, a single ribonucleo- Here we have shown through qualitative and quantitative POT1- tide in lieu of a deoxyribonucleotide at the fourth position of the ssDNA-RNA-binding experiments that a single thymidine (dT4) telomeric ssDNA dodecamer abrogates the ability of TPP1 to in the telomeric repeat contributes the most to hPOT1’s speci- increase the affinity of POT1 for this oligonucleotide. ficity for DNA. A previous study of RNA binding by S. pombe The lack of stimulation of hPOT1-4R binding in the presence POT1 revealed that the same dT4 residue as well as the preceding of TPP1 might arise from prevention of POT1-TPP1-N complex dT3 in the pombe telomeric sequence GGTTAC were most re- formation by the rU4 substitution. To ascertain whether TPP1-N sponsible for the specificity for DNA over RNA (14). Although forms a complex with POT1-4R, we carried out an EMSA of the absense of a 5-methyl is responsible for reduced binding of hPOT1-D120 and hPOT1-4R0.D120 and 4R0 differ from D12 rU4-containing oligonucleotides to S. pombe POT1, we find here 0 and 4R by the presence of 8 dTs at their 50 ends, which enhances that both the 2 OH and the lack of a 5-methyl group on rU4 in- the electrophoretic separation of the POT1-only and POT1-TPP1 terfere synergistically with mammalian POT1 binding. The pro- complexes. Fig. 4D shows that addition of hTPP1-N to tein used in the previous study consisted of only the first OB fold POT1-D120 and POT1-4R0 led to the formation of stable ternary of S. pombe POT1, and its nucleic acid binding characteristics complexes, as evidenced by the appearance of a slower migrating differ from that of the entire two-OB-fold DNA-binding domain band. Thus, TPP1-N remains bound to POT1 in the presence of (30, 31). However, we note that the TTA trinucleotide, which is the rU4 substitution but has lost its capacity to stabilize POT1 common to the two telomeric sequences, is bound very similarly binding to the 4R oligonucleotide. Interestingly, hTPP1 shows no by hPOT1 and the first OB fold of S. pombe POT1, with the same stimulation of POT1-oligonucleotide binding with 4D or 4–5D kind of dT-dT-Phe-stacking interactions and similar amino acid (Table 1). Put together, these data indicate that, although a dTat side chains involved in hydrogen bonding, so it seems likely that position 4 is important for high affinity of POT1-oligonucleotide this part of the S. pombe POT1–OB–nucleic acid interaction is binding, it is not sufficient to explain the mechanism by which correct. We conclude that the basis for POT1 discrimination TPP1 further enhances this affinity. We next probed the effect against RNA binding may be substantially conserved between of ribonucleotide substitutions at other positions on the stimu- fission yeast and mammals. lation of POT1-ssDNA binding by TPP1-N. We computed the We solved the structure of POT1 in complex with an oligonu- ratio of the KD of POT1-ssDNA-RNA complexes in the ab- cleotide with the rU4 substitution to provide a physical picture of sence and presence of TPP1-N [see “stimulation of binding by the deleterious effect of this substitution on POT1 binding. The hTPP1-N” (or mTPP1-N) in Table 1]. For D12, this ratio is 63 structure shows that the presence of rU4 causes a change in the with hPOT1 and 23 with mPOT1A. Upon inspection of Table 1, DNA conformation such that two hydrogen-bonding interactions it becomes immediately evident that stimulation of oligonucleo- of dT3 with POT1 are lost. In addition, the position of rU4 in the tide binding by POT1 in the presence of TPP1-N is weaker for all structure renders its polar O20 in close proximity to the hydropho- ribonucleotide-substituted oligonucleotides, although maximal bic region around Phe62 (Fig. 3A). It therefore seems possible BIOCHEMISTRY interference is seen in cases where there is a 4dT to 4rU change that burying the 20OH in a hydrophobic environment in the (4–6R and 4R in Table 1). Hence, the stimulation of POT1- rU4-containing complex leads to desolvation of this functional ssDNA binding by TPP1-N is extremely sensitive to the presence group, resulting in energy destabilization (32). Intriguingly, the of ribonucleotides throughout the telomeric sequence. only nucleotide where the C20 (and O20) is in a strictly hydropho- What is the consequence of the lack of stimulation of POT1- bic environment is rU4 (Fig. 3A). To test the idea that, in the oligonucleotide binding by TPP1-N on RNA discrimination? context of POT1, having a more hydrophobic Phe near a 20OH

Fig. 4. A single ribonucleotide substitution abrogates TPP1’s ability to stimulate POT1-ssDNA binding. Binding curves for mPOT1A-D12 (A), mPOT1A-4R (B), and hPOT1-4R (C) complexes in the presence (blue) and absence (red) of cognate TPP1-N proteins. Error bars represent the standard deviation of two independent measurements. (D) EMSA showing that hTPP1-N binds hPOT1-D120 and hPOT1-4R0 complexes to give distinct bands corresponding to hPOT1-hTPP1-N-D120 and hPOT1-hTPP1-N-4R0, respectively. Note that D120 and 4R0 contain eight deoxythymidines introduced at the 50 end of D12 and 4R sequences, respectively, to resolve the POT1-ssDNA versus POT1-TPP1-N-ssDNA complexes.

Nandakumar et al. PNAS ∣ January 12, 2010 ∣ vol. 107 ∣ no. 2 ∣ 655 Downloaded by guest on September 23, 2021 is less favorable than having a more hydrophilic Tyr, we probed DNA at replication forks and strongly discriminate against the effects of F62Y, Y89F, Y161F, and Y223F mutations on ssRNA (34, 35). It will be interesting to investigate how these pro- mPOT1A binding to ribonucleotide-substituted oligonucleotides. teins execute RNA discrimination. mPOT1A-F62Y showed merely a 2-fold binding defect with 4–6R as opposed to the 47-fold defect shown by the wild type. Y89F and Materials and Methods Y161F showed modest increases in binding defects for 4–6R and Protein Expression and Purification. hPOT1, hPOT1V2, hTPP1-N (aa 91–334), 7–9R, respectively, also consistent with the hypothesis that Phe mPOT1A (wild-type and mutants), and mTPP1-N (aa 1–246) proteins were ex- stacking discriminates against RNA binding and Tyr stacking pressed in bacteria and purified as detailed in SI Materials and Methods. permits RNA binding. The Phe-Tyr switch dictating DNA versus RNA specificity seen here has also been noted in T7 RNA Electrophoretic Mobility Shift Assays. Binding mixtures (10 μL) containing spe- ′ 32 polymerase, where the Y639F mutant relaxes RNA specificity by cified concentrations of 5 P-labeled oligonucleotides and POT1/TPP1 pro- allowing incorporation of dNTPs (33). teins were incubated at 4 °C and were then analyzed by electrophoresis through a nondenaturing 4–20% gradient polyacrylamide gel (Invitrogen). Another intriguing result that emerged from our binding Further details are provided in SI Materials and Methods. analysis is the fact that TPP1 binding greatly increases the DNA versus RNA discrimination of POT1. In the presence of TPP1-N, Filter-Binding Assays for KD Determination. Filter-binding experiments were the sensitivity of POT1-ssDNA binding to any ribonucleotide sub- performed in a 96-well dot blot apparatus. The protein-DNA mixtures were stitution in the binding sequence is greatly increased, suggesting incubated on ice and were then filtered through a precooled (at 4 °C) that TPP1 provides a layer of RNA discrimination above that pre- membrane sandwich containing a nitrocellulose membrane (BA85, What- sented by POT1 alone. Although the structure of the POT1-TPP1 man), a positively charged nylon membrane (Hybond Nþ, GE), and a filter complex has not been solved, there is a high-resolution crystal paper (Whatman). The membranes were air-dried and quantified by using structure of ssDNA bound to the structurally analogous O. nova a PhosphorImager. Further details are provided in SI Materials and Methods. TEBPα-β dimer (PDB: 1OTC) (12). In this structure, the α and β subunits form a groove that engulfs the ssDNA. If POT1- Structure Determination of hPOT1V2-dTrUd(AGGGTTAG) and hPOT1V2-D12. TPP1 sandwiches ssDNA in an analogous manner, the altered X-ray crystallography methods and solution of structures by molecular conformation of the sugar-phosphate backbone caused by replacement using PDB:1XJV as a starting model were performed as detailed ribo-substitution (Fig. 2D, E, and F) may result in the loss of in SI Materials and Methods. shape complementarity between the nucleic acid-POT1 surface and TPP1. Alternatively, the 20OH groups might directly perturb ACKNOWLEDGMENTS. We thank Corie Ralston from beam line 8.2.1 at the the TPP1–nucleic acid interface. Advanced Light Source, Berkeley, for help with x-ray diffraction data collection. We thank Dr. Sandy Chang (University of Texas, M.D. Anderson Finally, the issue of ssDNA versus RNA discrimination also Cancer Center) for providing a plasmid containing the cDNA sequence of occurs with single-stranded DNA binding protein (SSB) and re- mPOT1A. J.N. is a Howard Hughes Medical Institute fellow of the Helen plication protein A (RPA), proteins that bind single-stranded Hay Whitney Foundation.

1. Blackburn EH (2001) Switching and signaling at the telomere. Cell, 106:661–673. 20. Xin H, et al. (2007) TPP1 is a homologue of ciliate TEBP-beta and interacts with POT1 to 2. Makarov VL, Hirose Y, Langmore JP (1997) Long G tails at both ends of human recruit telomerase. Nature, 445:559–562. chromosomes suggest a C strand degradation mechanism for telomere shortening. 21. Wang F, et al. (2007) The POT1-TPP1 telomere complex is a telomerase processivity Cell, 88:657–666. factor. Nature, 445:506–510. 3. Rhodes D, Fairall L, Simonsson T, Court R, Chapman L (2002) Telomere architecture. 22. Azzalin CM, Reichenbach P, Khoriauli L, Giulotto E, Lingner J (2007) Telomeric repeat EMBO Rep, 3:1139–1145. containing RNA and RNA surveillance factors at mammalian chromosome ends. 4. Wright WE, Tesmer VM, Huffman KE, Levene SD, Shay JW (1997) Normal human chro- Science, 318:798–801. mosomes have long G-rich telomeric overhangs at one end. Dev, 11:2801–2809. 23. Luke B, Lingner J (2009) TERRA: Telomeric repeat-containing RNA. EMBO J, 5. de Lange T (2005) : The protein complex that shapes and safeguards human 28:2503–2510. – 0 0 telomeres. Genes Dev, 19:2100 2110. 24. Luke B, et al. (2008) The Rat1p 5 to 3 exonuclease degrades telomeric repeat- 6. Baumann P, Cech TR (2001) Pot1, the putative telomere end-binding protein in fission containing RNA and promotes telomere elongation in Saccharomyces cerevisiae. – yeast and humans. Science, 292:1171 1175. Mol Cell, 32:465–477. 7. Hockemeyer D, Daniels JP, Takai H, de Lange T (2006) Recent expansion of the 25. Wilkinson M (1988) A rapid and convenient method for isolation of nuclear, telomeric complex in rodents: Two distinct POT1 proteins protect mouse telomeres. cytoplasmic and total cellular RNA. Nucleic Acids Res, 16:10934. – Cell, 126:63 77. 26. Ishikawa F, Matunis MJ, Dreyfuss G, Cech TR (1993) Nuclear proteins that bind the 8. Baumann P, Podell E, Cech TR (2002) Human Pot1 (protection of telomeres) protein: pre-mRNA 30 splice site sequence r(UUAG/G) and the human telomeric DNA sequence Cytolocalization, structure, and alternative splicing. Mol Cell Biol, 22:8079–8087. d(TTAGGG)n. Mol Cell Biol, 13:4301–4310. 9. Wu L, et al. (2006) Pot1 deficiency initiates DNA damage checkpoint activation and 27. He H, et al. (2006) POT1b protects telomeres from end-to-end chromosomal fusions aberrant homologous recombination at telomeres. Cell, 126:49–62. and aberrant homologous recombination. EMBO J, 25:5180–5190. 10. Lei M, Podell ER, Cech TR (2004) Structure of human POT1 bound to telomeric 28. Palm W, Hockemeyer D, Kibe T, de Lange T (2009) Functional dissection of human and single-stranded DNA provides a model for chromosome end-protection. Nat Struct mouse POT1 proteins. Mol Cell Biol, 29:471–482. Mol Biol, 11:1223–1229. 29. Brunger AT, et al. (1998) Crystallography & NMR system: A new software suite for 11. Loayza D, De Lange T (2003) POT1 as a terminal transducer of TRF1 telomere length macromolecular structure determination. Acta Crystallogr, Sect D: Biol Crystallogr, control. Nature, 423:1013–1018. 54:905–921. 12. Horvath MP, Schweiker VL, Bevilacqua JM, Ruggles JA, Schultz SC (1998) Crystal 30. Croy JE, Podell ER, Wuttke DS (2006) A new model for Schizosaccharomyces structure of the Oxytricha nova telomere end binding protein complexed with single pombe telomere recognition: The telomeric single-stranded DNA-binding activity strand DNA. Cell, 95:963–974. – 13. Jacob NK, Lescasse R, Linger BR, Price CM (2007) Tetrahymena POT1a regulates of Pot11-389. J Mol Biol, 361:80 93. telomere length and prevents activation of a cell cycle checkpoint. Mol Cell Biol, 31. Trujillo KM, Bunch JT, Baumann P (2005) Extended DNA binding site in Pot1 broadens 27:1592–1601. sequence specificity to allow recognition of heterogeneous fission yeast telomeres. – 14. Lei M, Podell ER, Baumann P, Cech TR (2003) DNA self-recognition in the structure of J Biol Chem, 280:9119 9128. Pot1 bound to telomeric single-stranded DNA. Nature, 426:198–203. 32. Hansch C, Coats E (1970) Alpha-chymotrypsin: A case study of substituent constants 15. Shakirov EV, Surovtseva YV, Osbun N, Shippen DE (2005) The Arabidopsis Pot1 and and regression analysis in enzymic structure-activity relationships. J Pharm Sci, – Pot2 proteins function in telomere length homeostasis and chromosome end 59:731 743. protection. Mol Cell Biol, 25:7725–7733. 33. Sousa R, Padilla R (1995) A mutant T7 RNA polymerase as a DNA polymerase. EMBO J, 16. Tani A, Murata M (2005) Alternative splicing of Pot1 (protection of telomere)-like 14:4609–4621. genes in Arabidopsis thaliana. Genes Genet Syst, 80:41–48. 34. Curth U, Genschel J, Urbanke C, Greipel J (1996) In vitro and in vivo function of the 17. Surovtseva YV, et al. (2007) Arabidopsis POT1 associates with the telomerase RNP and C-terminus of Escherichia coli single-stranded DNA binding protein. Nucleic Acids Res, is required for telomere maintenance. EMBO J, 26:3653–3661. 24:2706–2711. 18. Ye JZ, et al. (2004) POT1-interacting protein PIP1: A telomere length regulator that 35. Kim C, Snyder RO, Wold MS (1992) Binding properties of replication protein A from recruits POT1 to the TIN2/TRF1 complex. Genes Dev, 18:1649–1654. human and yeast cells. Mol Cell Biol, 12:3050–3059. 19. Liu D, et al. (2004) PTOP interacts with POT1 and regulates its localization to 36. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of telomeres. Nat Cell Biol, 6:673–680. a protein. J Mol Biol, 157:105–132.

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