DNA Binding Features of Human POT1

The human telomeric protein POT1 is known to bind single-stranded telomeric DNA in vitro and to participate in the regulation of telomere maintenance by telomerase in vivo. We examined the in vitro DNA binding features of POT1. We report that deleting the oligosaccharide/oligonucleotide-binding fold of POT1 abrogates its DNA binding activity. The minimal binding site (MBS) for POT1 was found to be the telomeric nonamer 5′-TAGGGTTAG-3′, and the optimal substrate is [TTAGGG]n (n ≥ 2). POT1 displays exceptional sequence specificity when binding to MBS, tolerating changes only at position 7 (T7A). Whereas POT1 binding to MBS or [TTAGGG]2 was enhanced by the proximity of a 3′ end, POT1 was able to bind to a [TTAGGG]5 array when positioned internally. These data indicate that POT1 has a strong sequence preference for the human telomeric repeat tract and predict that POT1 can bind both the 3′ telomeric overhang and the displaced TTAGGG repeats at the base of the t-loop. The human telomeric protein POT1 is known to bind single-stranded telomeric DNA in vitro and to participate in the regulation of telomere maintenance by telomerase in vivo. We examined the in vitro DNA binding features of POT1. We report that deleting the oligosaccharide/oligonucleotide-binding fold of POT1 abrogates its DNA binding activity. The minimal binding site (MBS) for POT1 was found to be the telomeric nonamer 5′-TAGGGTTAG-3′, and the optimal substrate is [TTAGGG]n (n ≥ 2). POT1 displays exceptional sequence specificity when binding to MBS, tolerating changes only at position 7 (T7A). Whereas POT1 binding to MBS or [TTAGGG]2 was enhanced by the proximity of a 3′ end, POT1 was able to bind to a [TTAGGG]5 array when positioned internally. These data indicate that POT1 has a strong sequence preference for the human telomeric repeat tract and predict that POT1 can bind both the 3′ telomeric overhang and the displaced TTAGGG repeats at the base of the t-loop. Human telomeres are composed of 6-10 kbp of double-stranded TTAGGG repeats that end in a single-stranded overhang of several hundred nucleotides. The 3′ end of the telomeric overhang is the substrate of telomerase, the cellular reverse transcriptase that synthesizes telomeric repeats. The telomere can adopt a “t-loop” configuration, in which the 3′ overhang strand-invades the duplex (1Griffith J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. de Lange T. Cell. 1999; 97: 503-514Abstract Full Text Full Text PDF PubMed Scopus (1946) Google Scholar). The strand invasion results in a displacement loop (D-loop) of single-stranded TTAGGG repeats; these single-stranded repeats are not at a 3′ end. The actual 3′ end of the chromosome is predicted to be base-paired to the C-strand and therefore inaccessible to telomerase when telomeres are in the t-loop configuration. Proteins that bind to the double-stranded portion of human telomeres have been studied extensively (reviewed in Ref. 4Smogorzewska A. de Lange T. Annu. Rev. Biochem. 2004; (in press)PubMed Google Scholar). In particular, TRF1 binds as a dimer through a MYB-type helix-loop-helix domain. The MYB domains of TRF1 dimers bind to two double-stranded 5′-YTAGGGTTR-3′ half-sites independent of spacing or orientation (2Bianchi A. Stansel R.M. Fairall L. Griffith J.D. Rhodes D. de Lange T. EMBO J. 1999; 18: 5735-5744Crossref PubMed Scopus (169) Google Scholar, 3Bianchi A. Smith S. Chong L. Elias P. de Lange T. EMBO J. 1997; 16: 1785-1794Crossref PubMed Scopus (271) Google Scholar). Other proteins such as tankyrase 1 and 2, Tin2, PINX1, and POT1 are recruited to telomeres by TRF1 (4Smogorzewska A. de Lange T. Annu. Rev. Biochem. 2004; (in press)PubMed Google Scholar). The TRF1 complex is involved in the regulation of telomere length by cis-inhibition of telomerase. In telomerase-positive cells, the overexpression of TRF1 leads to telomere shortening, and the expression of a dominant negative form leads to telomere elongation (5van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1055) Google Scholar). The relationship between TRF1 and telomerase regulation at the 3′ telomere terminus is poorly understood. The human single-stranded telomeric DNA-binding protein POT1 was identified based on its sequence similarity to the Oxytricha nova TEBPα, known to be associated to the 16-base single-stranded telomeric extension in this organism (6Baumann P. Cech T.R. Science. 2001; 292: 1171-1175Crossref PubMed Scopus (810) Google Scholar). Orthologs in Schizosaccharomyces pombe, Arabidopsis, mouse, human, and other eukaryotes have been identified by homology to the N-terminal OB 1The abbreviations used are: OB fold, oligosaccharide/oligonucleotide-binding fold; MBS, minimal binding site; GST, glutathione S-transferase; RPA, replication protein A; nt, nucleotide(s). (oligosaccharide/oligonucleotide-binding) fold, a structural domain involved in DNA binding (7Baumann P. Podell E. Cech T.R. Mol. Cell. Biol. 2002; 22: 8079-8087Crossref PubMed Scopus (143) Google Scholar). The crystal structure of TEBPα revealed three OB folds, two involved in DNA binding and the third one necessary for a protein interaction with TEBPβ (8Horvath M.P. Schweiker V.L. Bevilacqua J.M. Ruggles J.A. Schultz S.C. Cell. 1998; 95: 963-974Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). TEBPα, -β, and the DNA form a ternary complex in which the α/β dimer forms a cage around the DNA, which itself is folded into a hairpin structure (8Horvath M.P. Schweiker V.L. Bevilacqua J.M. Ruggles J.A. Schultz S.C. Cell. 1998; 95: 963-974Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). No ortholog of TEBPβ has been found in mammals. In S. pombe, Pot1 (spPot1) is essential for the protection of chromosome ends (6Baumann P. Cech T.R. Science. 2001; 292: 1171-1175Crossref PubMed Scopus (810) Google Scholar). Deletion of the pot1+ gene leads to rapid telomere degradation. Survivors of this telomere loss have circularized chromosomes that lack all telomeric DNA. Binding studies have determined that spPot1 binds to a sequence representing fission yeast telomeric DNA (repeats of GGTTACA) but not to the human telomeric sequence (TTAGGG). The minimal binding site for the isolated DNA binding domain consists of six nucleotides, GGTTAC, with a binding constant of 83 nm (9Lei M. Baumann P. Cech T.R. Biochemistry. 2002; 41: 14560-14568Crossref PubMed Scopus (71) Google Scholar). Cooperative binding occurs on oligonucleotides containing multimeric sites, and spPot1 displays a preference for sequences close to a 3′ end. Taken together these data are consistent with a model in which the protein initially binds the very 3′ end of chromosomes and subsequently coats the entire telomeric overhang. Human POT1 has two important domains required for its function: an N-terminal OB fold, predicted to be necessary for DNA binding, and a protein interaction domain, mediating association with the TRF1 complex (10Loayza D. de Lange T. Nature. 2003; 424: 1013-1018Crossref PubMed Scopus (544) Google Scholar). Thus, POT1 is a good candidate for providing the link between TRF1 on the duplex telomeric portion and the 3′ overhang where telomerase acts. It is still unclear whether DNA binding is a primary event in POT1 targeting to telomeres or if it occurs after recruitment by TRF1. POT1 can be detected by immunofluorescence at telomeres, and can associate with telomeres through its interaction with the TRF1 complex (10Loayza D. de Lange T. Nature. 2003; 424: 1013-1018Crossref PubMed Scopus (544) Google Scholar). The exact role of the DNA binding activity of POT1 is unknown, but the expression of a mutant form of POT1 missing the OB fold (POT1ΔOB) leads to extensive telomere elongation in telomerase-positive cells (10Loayza D. de Lange T. Nature. 2003; 424: 1013-1018Crossref PubMed Scopus (544) Google Scholar). The simplest model is that POT1 lies downstream of the TRF1 complex in the telomere length regulation pathway. POT1 is proposed to inhibit telomerase in cis at chromosome ends in response to the length of the duplex portion and as such could play a role in the counting mechanism performed by the TRF1 complex. POT1 has also been implicated as a positive factor for telomere length control. In some settings, overexpression of full-length POT1 (with short N-terminal and C-terminal extensions) was found to lead to telomere elongation (11Colgin L.M. Baran K. Baumann P. Cech T.R. Reddel R.R. Curr. Biol. 2003; 13: 942-946Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). It is possible that POT1 has a dual mode of function at telomeres; one would be inhibitory to telomerase, and the other promoting telomere elongation. Such a dual role in telomere length regulation has been proposed for the distantly related protein Cdc13p in Saccharomyces cerevisiae (12Chandra A. Hughes T.R. Nugent C.I. Lundblad V. Genes Dev. 2001; 15: 404-414Crossref PubMed Scopus (191) Google Scholar). The main form of POT1 detected by Western blotting is the full-length protein (10Loayza D. de Lange T. Nature. 2003; 424: 1013-1018Crossref PubMed Scopus (544) Google Scholar). However, POT1 transcripts are subject to alternative splicing, possibly leading to the expression of C-terminal truncations in certain cell types (7Baumann P. Podell E. Cech T.R. Mol. Cell. Biol. 2002; 22: 8079-8087Crossref PubMed Scopus (143) Google Scholar). In vitro, one of the variants (variant 2), corresponding to the N-terminal 38 kDa of the protein, displays an 8-fold higher binding affinity than full-length POT1. It is not known whether the alternatively spliced transcripts yield stable proteins or what the role of these variants in telomere biology might be. Human POT1 binds to single-stranded human telomeric TTAGGG repeats but not to double-stranded telomeric DNA nor to the C-rich telomeric repeat strand (6Baumann P. Cech T.R. Science. 2001; 292: 1171-1175Crossref PubMed Scopus (810) Google Scholar). POT1 only binds efficiently to the human telomeric sequence and not to the S. pombe telomeric DNA nor to the O. nova TTTTGGGG sequence (6Baumann P. Cech T.R. Science. 2001; 292: 1171-1175Crossref PubMed Scopus (810) Google Scholar). To date, the minimal binding site, sequence specificity, and 3′ end dependence of POT1 have not been studied in detail. Here, we address these and other aspects of the DNA binding features of POT1. Purification of Escherichia coli or Baculovirus-expressed POT1—The cloning of the POT1 and POT1ΔOB cDNAs was described previously (10Loayza D. de Lange T. Nature. 2003; 424: 1013-1018Crossref PubMed Scopus (544) Google Scholar). The full-length POT1 cDNA was cloned as a BamHI-XhoI fragment in FastBac HTb (Clontech), adding a His6 tag to the N terminus and the transfections, and virus amplifications and protein production were performed as described in the manufacturer's protocols. The protein was purified out of 100 ml of Sf21 cells 48 h after infection (m.o.i. = 5). For protein purification from E. coli, the POT1 and POT1ΔOB cDNAs were cloned in the BamHI and XhoI sites of pGEX-4T2 (Amersham Biosciences), resulting in N-terminal GST fusions. GST fusion proteins were purified on glutathione beads as directed by the manufacturer. After purification, the protein was dialyzed against 20 mm Hepes, pH 7.9, 500 mm KCl (150 mm for GST fusions), and 20% glycerol, flash-frozen in liquid nitrogen, and stored in aliquots at -80 °C. The binding affinity of POT1 declined 3-5-fold over a period of 2 weeks and was stable afterward. Oligonucleotides and Probe Labeling—All oligonucleotides were obtained from Genelink as gel-purified 50-mers with indicated random and telomeric sequences (see Table I). Oligonucleotides were labeled at the 5′ end with T4 polynucleotide kinase (New England Biolabs) and [γ-32P]ATP (3000 Ci/mmol, PerkinElmer Life Sciences) and purified through a Sephadex G50 column in 10 mm Tris-Hcl, pH 7.5, 100 mm NaCl, 1 mm EDTA, 0.1% SDS. The labeled oligos were extracted with phenol/chloroform/isoamyl alcohol, precipitated with 0.2 m NaAc, pH 5.5, and 2 volumes of EtOH (-20 °C overnight) and dissolved in 10 mm Tris, pH 8.0.Table ISequences of POT1 binding substratesNameSequence (5′ to 3′)aNon-telomeric portions of the oligonucleotides are: A, GCAAGCT-TTA; B, CCGATACAGC; C, CGCTCA; D, GACTGA; E, CTCGAA; F, GAACTC; G, CAC; H, GAACTCGATCC; J, CACTGCAACT; K, GAACTCGAC; L, CACTGCAAC.tel-1A-B-C-D-E-F- (TTAGGG)tel-2A-B-C-D-E- (TTAGGG)2tel-3A-B-C-D- (TTAGGG)3tel-4A-B-C- (TTAGGG)4tel-5A-B- (TTAGGG)5int-5A- (TTAGGG)5-Bint-2A-K- (TTAGGG)2-L-Bint-MBSA-H- (TAGGGTTAG) -J-B5′-5(TTAGGG)5-A-BMBSA-B-C-D-E-G-TAGGGTTAGMBS+1A-B-C-D-E-CCTTAGGGTTAGPermut1A-B-C-D-E-G-AGGGTTAGGPermut2A-B-C-D-E-G-GGGTTAGGGPermut3A-B-C-D-E-G-GGTTAGGGTPermut4A-B-C-D-E-G-GTTAGGGTTPermut5A-B-C-D-E-G-TTAGGGTTAMBS-T1AA-B-C-D-E-G-CAGGGTTAGMBS-A2TA-B-C-D-E-G-TTGGGTTAGMBS-G3CA-B-C-D-E-G-TACGGTTAGMBS-G4CA-B-C-D-E-G-TAGCGTTAGMBS-G5CA-B-C-D-E-G-TAGGCTTAGMBS-T6AA-B-C-D-E-G-TAGGGATAGMBS-T7AA-B-C-D-E-G-TAGGGTAAGMBS-A8TA-B-C-D-E-G-TAGGGTTTGMBS-G9CA-B-C-D-E-G-TAGGGTTACMBS-G9AA-B-C-D-E-G-TAGGGTTAAa Non-telomeric portions of the oligonucleotides are: A, GCAAGCT-TTA; B, CCGATACAGC; C, CGCTCA; D, GACTGA; E, CTCGAA; F, GAACTC; G, CAC; H, GAACTCGATCC; J, CACTGCAACT; K, GAACTCGAC; L, CACTGCAAC. Open table in a new tab Band Shift Assays—The binding reactions were performed in 20 μl of the following buffer: 20 mm glycine-NaOH, pH 9.0, 0.1 mm dithiothreitol, 2% glycerol, 50 ng of β-casein, 0.5 μg of sonicated and denatured E. coli DNA (mean size ∼400 nt), and 0.25 nm probe. The protein was added last, and the binding reaction was incubated for 30 min at room temperature. For direct binding, a range of 0.1-5 μg of protein was used. For competition assays, 0.5-1 μg of protein was added to the reactions containing up to 100-fold molar excess of unlabeled competitor oligonucleotide. Electrophoresis was performed in 0.6% agarose gels run in 0.1× Tris borate EDTA. The gels were run for 40 min at 160 V, dried on Whatman DE81 paper at 80 °C and exposed on phosphorimaging screens. Quantitation was performed the Purification of POT1 and Band Shift was in cells or in E. coli and purified on affinity and Band were performed with single-stranded DNA I). binding were at a constant length of 50 by the length of the random sequence at the 5′ end of the to for in the length of the telomeric POT1 not bind to of the random (see for POT1 binding to TTAGGG repeat were and optimal binding at pH in the of and 0.5 μg of single-stranded E. coli DNA competitor not were incubated for 30 min at room and on 0.6% agarose gels in 0.1× Tris borate (see or was able to a containing or TTAGGG repeats and C, and For of protein, the was as as 20 nm as determined from the of protein required to bind of the probe. To whether the N-terminal OB fold was required for the in vitro DNA binding activity of we a protein (POT1ΔOB) the of the POT1 the that up the OB mutant DNA binding activity and protein we that POT1ΔOB was at than the full-length POT1 protein and data not that the N terminus of POT1 is essential for DNA binding in No binding was detected with POT1ΔOB on two or TTAGGG repeats. POT1 [TTAGGG]n ≥ 2 but a TTAGGG at a 3′ we found that POT1 with two or TTAGGG repeats at a 3′ end or a with only one TTAGGG sequence was not 2). was obtained both by direct binding and by the competition of for the binding of POT1 to 2, and Table Thus, the minimal binding site for POT1 is than and than or to of POT1 for position of short but not sequences are by telomeric repeats by binding was with 0.1-5 μg of POT1 were with as a probe. The are from Open table in a new tab a sequences are by telomeric repeats by binding was with 0.1-5 μg of POT1 were with as a probe. The are from the to its the telomeric POT1 binding In a only the portion in double-stranded form as as the single-stranded These are consistent with previously results (6Baumann P. Cech T.R. Science. 2001; 292: 1171-1175Crossref PubMed Scopus (810) Google that POT1 binds only to single-stranded telomeric We subsequently used competition for POT1 binding to to the of POT1 to with We the telomeric repeats in the by random sequences (see Table I). with repeats efficiently for POT1 binding to The competition as by the molar excess of the competitor required for in POT1 binding to the labeled was when the telomeric repeats were at the 3′ end of the binding substrate or However, with containing only two was a when the telomeric sequence was containing two TTAGGG not for POT1 binding to the itself the TTAGGG repeats at the 3′ the competition in binding of molar excess of was the on short POT1 displays a preference for binding sites at a 3′ end than a 100-fold for with two TTAGGG preference for the 3′ end was detected with containing TTAGGG repeats 2, and we that a 3′ end is not required for POT1 binding, but the binding to short sites is when are at a 3′ end. the TTAGGG repeats at the 5′ end not the binding as a substrate with TTAGGG repeats at the 5′ end for binding to the excess = The POT1 Binding nonamer binding site for POT1 was identified by oligonucleotides with of one and a TTAGGG repeats positioned at a 3′ end all possible only one sequence was of efficiently for POT1 Table The molar excess required for competition was than for itself and binding the binding of POT1 to the sequence and the lack of binding to other the of the site its to for by than (see Table In the of one 5′ in not binding as by direct binding not These data indicated that the sequence the minimal binding site (MBS) sequence for POT1. direct binding, was in affinity between and MBS binds to a of a telomeric sequences are by and telomeric repeats by binding was with 0.1-5 μg of POT1 were with as a probe. The are from not Open table in a new tab a sequences are by and telomeric repeats by binding was with 0.1-5 μg of POT1 were with as a probe. The are from not To whether POT1 a 3′ end to bind to MBS, we this sequence in a 5′ and 3′ by random was the for the with two TTAGGG POT1 not bind to the MBS by direct binding or by competition Thus, the lack of binding to short sequences at sites is not to the used. We that the sequence the minimal binding for POT1 when at the 3′ end of the DNA. POT1 with minimal binding site a for the of the sequence specificity of POT1. or were at of the in MBS, and the resulting MBS variants were by competition for POT1 binding to Table affinity POT1 binding with only one MBS the sequence a of to at position 7 to as Table on competition assays, POT1 to with an affinity than to MBS excess of for with for MBS, Table direct binding, POT1 also to MBS and The sequence containing a to at position (in was also but with a loss of affinity with MBS of the other changes the binding of POT1 to MBS and Table specificity of POT1 when binding to binding was with 0.1-5 μg of POT1 excess at were with as a probe. The are binding was with 0.1-5 μg of POT1 were with as a probe. The are Open table in a new tab a for the of the sequence specificity by POT1 when binding to MBS, we the binding of POT1 to repeats of the sequence GGTTAC, a that binding based on the lack of binding of POT1 to and and with the results of the MBS the substrate containing repeats could not with for POT1 binding However, a DNA substrate with the only in the repeat in was an competitor the binding activity of POT1 and that changes in the telomeric repeat at binding sites to binding activity. We that POT1 displays sequence specificity in the of the minimal binding The of the DNA binding of POT1 is important for its function at In this we have the minimal binding site for human POT1 and determined the important for binding to this The sequence the only nonamer that POT1 with affinity to telomeric repeat The sequence its affinity for a preference for 3′ end binding on short However, human and telomeres have been to of TTAGGG which can MBS to POT1 binds very to such TTAGGG repeat when are not at a 3′ that of the 3′ overhang and of the telomeric could be by POT1. The POT1 site is at in which is that OB with short sites is that POT1 binds as a dimer or OB of the OB lack strong primary sequence it is not that POT1 two or three of these is that POT1 to which with an site a OB fold T.R. M. Lundblad V. S. A. 97: PubMed Scopus Google Scholar, R.M. Hughes T.R. Lundblad V. Science. 2002; PubMed Scopus Google Scholar). In this the 5′ sequence a of binding in by a the binding substrate to R.M. Hughes T.R. Lundblad V. Science. 2002; PubMed Scopus Google Scholar, Biochemistry. 2003; PubMed Scopus Google Scholar). is that human POT1 the telomeric site through both protein DNA and a mode of substrate by the complex (8Horvath M.P. Schweiker V.L. Bevilacqua J.M. Ruggles J.A. Schultz S.C. Cell. 1998; 95: 963-974Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The in the complex could and higher of the single-stranded DNA. Thus, the MBS sequence could be a good POT1 substrate both it the optimal base and and has the to adopt the for POT1 The of the crystal structure of human POT1 to the DNA on these the role of the 3′ terminus in the of the complex could as in the which could the preference of POT1 for its minimal binding site at a 3′ end. We have previously that an N-terminal which the DNA binding domain, can still to telomeres (10Loayza D. de Lange T. Nature. 2003; 424: 1013-1018Crossref PubMed Scopus (544) Google Scholar). that the DNA binding function is not necessary for targeting POT1 to telomeres POT1 is recruited to telomeres by protein interaction through the TRF1 it could associate with its DNA binding site as a the of POT1 at chromosome ends would on two its interaction with the TRF1 complex and its to In this of the 3′ which is a third of the not be an important POT1 between MBS and sites that have of POT1 to between telomeric repeats and related sequences could be to its function in sequences telomeric DNA at chromosome sites J. PubMed Scopus Google Scholar). In some these sequences are a of DNA Nature. PubMed Scopus Google Scholar). The sequence specificity of POT1 be to binding to these repeats. is important the TRF1 complex can be on the repeats A. Steensel B. Bianchi A. S. de Lange T. Mol. Cell. Biol. PubMed Scopus Google POT1 to these Thus, the sequence specificity by POT1 not be important for actual targeting and of the protein to telomeres but be important for POT1 from binding to sites in the In this it would be to the of POT1 and replication protein the single-stranded DNA-binding protein involved in DNA replication Annu. Rev. Biochem. 1997; PubMed Scopus Google to telomeric and It is that the affinity of for the telomeric sequence is than that of which would in the of by POT1 from the telomeric overhang. the other not sequence specificity on single-stranded DNA, it could POT1 from sites, which be important DNA In would have to POT1 from telomeric DNA the replication of It is that function to this We the de Lange for on the and D. for