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DNA Binding Features of Human POT1 a NONAMER 5Ј-TAGGGTTAG-3Ј MINIMAL BINDING SITE, SEQUENCE SPECIFICITY, and INTERNAL BINDING to MULTIMERIC SITES*

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DNA Binding Features of Human POT1 a NONAMER 5Ј-TAGGGTTAG-3Ј MINIMAL BINDING SITE, SEQUENCE SPECIFICITY, and INTERNAL BINDING to MULTIMERIC SITES* THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 13, Issue of March 26, pp. 13241–13248, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. DNA Binding Features of Human POT1 A NONAMER 5Ј-TAGGGTTAG-3Ј MINIMAL BINDING SITE, SEQUENCE SPECIFICITY, AND INTERNAL BINDING TO MULTIMERIC SITES* Received for publication, November 10, 2003, and in revised form, January 7, 2004 Published, JBC Papers in Press, January 7, 2004, DOI 10.1074/jbc.M312309200 Diego Loayza‡§, Heather Parsons‡, Jill Donigian, Kristina Hoke¶, and Titia de Langeʈ From the Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, New York 10021 The human telomeric protein POT1 is known to bind tankyrase 1 and 2, Tin2, PINX1, and POT1 are recruited to single-stranded telomeric DNA in vitro and to partici- telomeres by TRF1 (4). The TRF1 complex is involved in the pate in the regulation of telomere maintenance by te- regulation of telomere length by cis-inhibition of telomerase. In lomerase in vivo. We examined the in vitro DNA binding telomerase-positive cells, the overexpression of TRF1 leads to features of POT1. We report that deleting the oligosac- telomere shortening, and the expression of a dominant nega- charide/oligonucleotide-binding fold of POT1 abrogates tive form leads to telomere elongation (5). The relationship its DNA binding activity. The minimal binding site between TRF1 and telomerase regulation at the 3Ј telomere (MBS) for POT1 was found to be the telomeric nonamer terminus is poorly understood. ؅ ؅ 5 -TAGGGTTAG-3 , and the optimal substrate is The human single-stranded telomeric DNA-binding protein [TTAGGG]n (n > 2). POT1 displays exceptional sequence POT1 was identified based on its sequence similarity to the specificity when binding to MBS, tolerating changes Oxytricha nova TEBP␣, known to be associated to the 16-base only at position 7 (T7A). Whereas POT1 binding to MBS single-stranded telomeric extension in this organism (6). Or- or [TTAGGG] was enhanced by the proximity of a 3؅ 2 thologs in Schizosaccharomyces pombe, Arabidopsis, mouse, end, POT1 was able to bind to a [TTAGGG] array when 5 human, and other eukaryotes have been identified by homology positioned internally. These data indicate that POT1 has 1 a strong sequence preference for the human telomeric to the N-terminal OB (oligosaccharide/oligonucleotide-bind- repeat tract and predict that POT1 can bind both the 3؅ ing) fold, a structural domain involved in DNA binding (7). The telomeric overhang and the displaced TTAGGG repeats crystal structure of TEBP␣ revealed three OB folds, two in- at the base of the t-loop. volved in DNA binding and the third one necessary for a pro- tein interaction with TEBP␤ (8). TEBP␣,-␤, and the DNA form a ternary complex in which the ␣/␤ dimer forms a cage around Human telomeres are composed of 6–10 kbp of double- the DNA, which itself is folded into a hairpin structure (8). No stranded TTAGGG repeats that end in a single-stranded over- ortholog of TEBP␤ has been found in mammals. hang of several hundred nucleotides. The 3Ј end of the telo- In S. pombe, Pot1 (spPot1) is essential for the protection of meric overhang is the substrate of telomerase, the cellular chromosome ends (6). Deletion of the pot1ϩ gene leads to rapid reverse transcriptase that synthesizes telomeric repeats. The telomere degradation. Survivors of this telomere loss have cir- telomere can adopt a “t-loop” configuration, in which the 3Ј cularized chromosomes that lack all telomeric DNA. Binding overhang strand-invades the duplex (1). The strand invasion studies have determined that spPot1 binds to a sequence rep- results in a displacement loop (D-loop) of single-stranded resenting fission yeast telomeric DNA (repeats of GGTTACA) TTAGGG repeats; these single-stranded repeats are not at a 3Ј but not to the human telomeric sequence (TTAGGG). The min- end. The actual 3Ј end of the chromosome is predicted to be imal binding site for the isolated DNA binding domain consists base-paired to the C-strand and therefore inaccessible to telom- of six nucleotides, GGTTAC, with a binding constant of 83 nM erase when telomeres are in the t-loop configuration. (9). Cooperative binding occurs on oligonucleotides containing Proteins that bind to the double-stranded portion of human multimeric sites, and spPot1 displays a preference for se- telomeres have been studied extensively (reviewed in Ref. 4). In quences close to a 3Ј end. Taken together these data are con- particular, TRF1 binds as a dimer through a MYB-type helix- sistent with a model in which the protein initially binds the loop-helix domain. The MYB domains of TRF1 dimers bind to very 3Ј end of chromosomes and subsequently coats the entire two double-stranded 5Ј-YTAGGGTTR-3Ј half-sites independ- telomeric overhang. ent of spacing or orientation (2, 3). Other proteins such as 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 * This work is supported in part by Grants CA076027 and T32 CA09673–26 from the NCI, National Institutes of Health. The costs of association with the TRF1 complex (10). Thus, POT1 is a good publication of this article were defrayed in part by the payment of page candidate for providing the link between TRF1 on the duplex charges. This article must therefore be hereby marked “advertisement” telomeric portion and the 3Ј overhang where telomerase acts. It in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. is still unclear whether DNA binding is a primary event in ‡ These authors contributed equally to this study. § Recipient of an Ann Siegel postdoctoral fellowship from the Amer- POT1 targeting to telomeres or if it occurs after recruitment by ican Cancer Society (PF-00-135-01). TRF1. ¶ Supported by National Institutes of Health Medical Scientist Train- POT1 can be detected by immunofluorescence at telomeres, ing Program Grant GM07739 to the Cornell/Rockefeller/Sloan-Ketter- and can associate with telomeres through its interaction with ing Tri-Institutional M.D.-Ph.D. Program. ʈ To whom correspondence should be addressed: Laboratory for Cell Biology and Genetics, The Rockefeller University, 1230 York Ave, New 1 The abbreviations used are: OB fold, oligosaccharide/oligonucleo- York, NY 10021. Tel.: 212-327-8146; Fax: 212-327-7147; E-mail: tide-binding fold; MBS, minimal binding site; GST, glutathione S-trans- [email protected]. ferase; RPA, replication protein A; nt, nucleotide(s). This paper is available on line at http://www.jbc.org 13241 13242 POT1 Binding Site FIG.1.The OB fold of POT1 is nec- essary for DNA binding. A, purification of Bac-His6-POT1 from Sf21 cells (left) and GST-POT1 (middle) and GST- POT1⌬OB (right) from E. coli. The elution (E), input (IN), and flow-through (FT) fractions were separated by SDS-PAGE and stained with Coomassie Blue. B, di- rect binding of Bac-His6-POT1 to an oli- gonucleotide probe with three single- stranded telomeric repeats without random sequence. Here and below, each TTAGGG repeat is represented by an ar- rowhead. C and D, deletion of the N-ter- minal OB fold abrogates binding. The POT1⌬OB truncation removes 126 amino acids from the N terminus. The probes in C and D contained 2 or 5 copies of TTAGGG at the 3Ј end of a 50-mer (tel-2 and tel-5, respectively; see Table I). the TRF1 complex (10). The exact role of the DNA binding telomere elongation in telomerase-positive cells (10). The sim- activity of POT1 is unknown, but the expression of a mutant plest model is that POT1 lies downstream of the TRF1 complex form of POT1 missing the OB fold (POT1⌬OB) leads to extensive in the telomere length regulation pathway. POT1 is proposed to POT1 Binding Site 13243 inhibit telomerase in cis at chromosome ends in response to the TABLE I length of the duplex portion and as such could play a role in the Sequences of POT1 binding substrates counting mechanism performed by the TRF1 complex. POT1 Name Sequence (5Ј to 3Ј)a has also been implicated as a positive factor for telomere length tel-1 A-B-C-D-E-F-(TTAGGG) control. In some settings, overexpression of full-length POT1 tel-2 A-B-C-D-E-(TTAGGG)2 (with short N-terminal and C-terminal extensions) was found tel-3 A-B-C-D-(TTAGGG)3 tel-4 A-B-C-(TTAGGG) to lead to telomere elongation (11). It is possible that POT1 4 tel-5 A-B-(TTAGGG) has a dual mode of function at telomeres; one would be 5 int-5 A-(TTAGGG)5-B inhibitory to telomerase, and the other promoting telomere int-2 A-K-(TTAGGG)2-L-B elongation. Such a dual role in telomere length regulation int-MBS A-H-(TAGGGTTAG)-J-B 5Ј-5 (TTAGGG) -A-B has been proposed for the distantly related protein Cdc13p in 5 MBS A-B-C-D-E-G-TAGGGTTAG Saccharomyces cerevisiae (12). MBSϩ1 A-B-C-D-E-CCTTAGGGTTAG The main form of POT1 detected by Western blotting is the Permut1 A-B-C-D-E-G-AGGGTTAGG full-length protein (10). However, POT1 transcripts are subject Permut2 A-B-C-D-E-G-GGGTTAGGG Permut3 A-B-C-D-E-G-GGTTAGGGT to alternative splicing, possibly leading to the expression of Permut4 A-B-C-D-E-G-GTTAGGGTT C-terminal truncations in certain cell types (7). In vitro, one of Permut5 A-B-C-D-E-G-TTAGGGTTA the variants (variant 2), corresponding to the N-terminal 38 MBS-T1A A-B-C-D-E-G-CAGGGTTAG kDa of the protein, displays an 8-fold higher binding affinity MBS-A2T A-B-C-D-E-G-TTGGGTTAG MBS-G3C A-B-C-D-E-G-TACGGTTAG than full-length POT1.
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