HOT1 is a mammalian direct telomere repeat-binding protein contributing to telomerase recruitment
2013; Springer Nature; Volume: 32; Issue: 12 Linguagem: Inglês
10.1038/emboj.2013.105
ISSN1460-2075
AutoresDennis Kappei, Falk Butter, Christian Benda, Marion Scheibe, Irena Drašković, Michelle Stevense, Clara Lopes Novo, Claire Basquin, Masatake Araki, Kimi Araki, Dragomir B. Krastev, Ralf Kittler, Rolf Jessberger, Arturo Londoño‐Vallejo, Matthias Mann, Frank Buchholz,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoArticle17 May 2013Open Access Source Data HOT1 is a mammalian direct telomere repeat-binding protein contributing to telomerase recruitment Dennis Kappei Dennis Kappei Medical Systems Biology, Faculty of Medicine Carl Gustav Carus, University Cancer Center, Dresden University of Technology, 01307, Dresden, Germany Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Falk Butter Falk Butter Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Christian Benda Christian Benda Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Marion Scheibe Marion Scheibe Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Irena Draškovič Irena Draškovič Telomeres & Cancer Laboratory, labellisé LIGUE, UMR3244, Institut Curie-CNRS-UPMC, Paris, France Search for more papers by this author Michelle Stevense Michelle Stevense Institute of Physiological Chemistry, Faculty of Medicine Carl Gustav Carus, Dresden University of Technology, Dresden, Germany Search for more papers by this author Clara Lopes Novo Clara Lopes Novo Telomeres & Cancer Laboratory, labellisé LIGUE, UMR3244, Institut Curie-CNRS-UPMC, Paris, France Search for more papers by this author Claire Basquin Claire Basquin Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Masatake Araki Masatake Araki Institute of Resource Development and Analysis, Kumamoto University, Honjo, Japan Search for more papers by this author Kimi Araki Kimi Araki Institute of Resource Development and Analysis, Kumamoto University, Honjo, Japan Search for more papers by this author Dragomir Blazhev Krastev Dragomir Blazhev Krastev Medical Systems Biology, Faculty of Medicine Carl Gustav Carus, University Cancer Center, Dresden University of Technology, 01307, Dresden, Germany Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Ralf Kittler Ralf Kittler Eugene McDermott Center for Human Growth and Development, UT Southwestern Medical Center at Dallas, Dallas, TX, USA Search for more papers by this author Rolf Jessberger Rolf Jessberger Institute of Physiological Chemistry, Faculty of Medicine Carl Gustav Carus, Dresden University of Technology, Dresden, Germany Search for more papers by this author J Arturo Londoño-Vallejo J Arturo Londoño-Vallejo Telomeres & Cancer Laboratory, labellisé LIGUE, UMR3244, Institut Curie-CNRS-UPMC, Paris, France Search for more papers by this author Matthias Mann Corresponding Author Matthias Mann Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Frank Buchholz Corresponding Author Frank Buchholz Medical Systems Biology, Faculty of Medicine Carl Gustav Carus, University Cancer Center, Dresden University of Technology, 01307, Dresden, Germany Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Dennis Kappei Dennis Kappei Medical Systems Biology, Faculty of Medicine Carl Gustav Carus, University Cancer Center, Dresden University of Technology, 01307, Dresden, Germany Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Falk Butter Falk Butter Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Christian Benda Christian Benda Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Marion Scheibe Marion Scheibe Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Irena Draškovič Irena Draškovič Telomeres & Cancer Laboratory, labellisé LIGUE, UMR3244, Institut Curie-CNRS-UPMC, Paris, France Search for more papers by this author Michelle Stevense Michelle Stevense Institute of Physiological Chemistry, Faculty of Medicine Carl Gustav Carus, Dresden University of Technology, Dresden, Germany Search for more papers by this author Clara Lopes Novo Clara Lopes Novo Telomeres & Cancer Laboratory, labellisé LIGUE, UMR3244, Institut Curie-CNRS-UPMC, Paris, France Search for more papers by this author Claire Basquin Claire Basquin Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Masatake Araki Masatake Araki Institute of Resource Development and Analysis, Kumamoto University, Honjo, Japan Search for more papers by this author Kimi Araki Kimi Araki Institute of Resource Development and Analysis, Kumamoto University, Honjo, Japan Search for more papers by this author Dragomir Blazhev Krastev Dragomir Blazhev Krastev Medical Systems Biology, Faculty of Medicine Carl Gustav Carus, University Cancer Center, Dresden University of Technology, 01307, Dresden, Germany Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Ralf Kittler Ralf Kittler Eugene McDermott Center for Human Growth and Development, UT Southwestern Medical Center at Dallas, Dallas, TX, USA Search for more papers by this author Rolf Jessberger Rolf Jessberger Institute of Physiological Chemistry, Faculty of Medicine Carl Gustav Carus, Dresden University of Technology, Dresden, Germany Search for more papers by this author J Arturo Londoño-Vallejo J Arturo Londoño-Vallejo Telomeres & Cancer Laboratory, labellisé LIGUE, UMR3244, Institut Curie-CNRS-UPMC, Paris, France Search for more papers by this author Matthias Mann Corresponding Author Matthias Mann Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Frank Buchholz Corresponding Author Frank Buchholz Medical Systems Biology, Faculty of Medicine Carl Gustav Carus, University Cancer Center, Dresden University of Technology, 01307, Dresden, Germany Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Author Information Dennis Kappei1,2,‡, Falk Butter3,‡, Christian Benda4, Marion Scheibe3, Irena Draškovič5, Michelle Stevense6, Clara Lopes Novo5, Claire Basquin4, Masatake Araki7, Kimi Araki7, Dragomir Blazhev Krastev1,2, Ralf Kittler8, Rolf Jessberger6, J Arturo Londoño-Vallejo5, Matthias Mann 3 and Frank Buchholz 1,2 1Medical Systems Biology, Faculty of Medicine Carl Gustav Carus, University Cancer Center, Dresden University of Technology, 01307, Dresden, Germany 2Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 3Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany 4Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany 5Telomeres & Cancer Laboratory, labellisé LIGUE, UMR3244, Institut Curie-CNRS-UPMC, Paris, France 6Institute of Physiological Chemistry, Faculty of Medicine Carl Gustav Carus, Dresden University of Technology, Dresden, Germany 7Institute of Resource Development and Analysis, Kumamoto University, Honjo, Japan 8Eugene McDermott Center for Human Growth and Development, UT Southwestern Medical Center at Dallas, Dallas, TX, USA ‡These authors contributed equally to this work. *Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. Tel.:+49 89 8578 2557; Fax:+49 89 8578 2219; E-mail:[email protected] or Medical Systems Biology, University Hospital and Medical Faculty Carl Gustav Carus, University of Technology Dresden, Fetscherstrasse 74, 01307 Dresden, Germany. Tel.:+49 351 46340288; Fax:+49 351 46340289; E-mail: [email protected] The EMBO Journal (2013)32:1681-1701https://doi.org/10.1038/emboj.2013.105 There is a Have you seen? (June 2013) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the telomerase complex to telomeres through a not yet fully understood mechanism. Factors promoting telomerase–telomere interaction are expected to directly bind telomeres and physically interact with the telomerase complex. In search for such a factor we carried out a SILAC-based DNA–protein interaction screen and identified HMBOX1, hereafter referred to as homeobox telomere-binding protein 1 (HOT1). HOT1 directly and specifically binds double-stranded telomere repeats, with the in vivo association correlating with binding to actively processed telomeres. Depletion and overexpression experiments classify HOT1 as a positive regulator of telomere length. Furthermore, immunoprecipitation and cell fractionation analyses show that HOT1 associates with the active telomerase complex and promotes chromatin association of telomerase. Collectively, these findings suggest that HOT1 supports telomerase-dependent telomere elongation. Introduction Telomeres, the nucleoprotein structures at the ends of chromosomes, consist of 5′-TTAGGG-3′ repeats bound by a dedicated set of proteins forming the shelterin complex (Palm and de Lange, 2008). Three members of the six protein complex directly bind telomeric DNA and were the only direct telomere-specific binding proteins known so far: TRF1 and TRF2 bind double-stranded DNA (dsDNA; Zhong et al, 1992; Bilaud et al, 1997; Broccoli et al, 1997), whereas POT1 binds single-stranded 5′-TTAGGG-3′ repeats (Baumann, 2001). This complex constitutively associates with telomeres and shields the ends of linear chromosomes from being recognized as a double-stranded break, thus protecting telomeres from end-to-end fusions (Palm and de Lange, 2008). While this solves the end-protection problem (de Lange, 2009), maintaining telomere integrity itself is of outstanding importance and major factors may have remained elusive. Telomere length homeostasis, a crucial process in stem cell biology, aging and cancer, depends on the equilibrium between telomere lengthening (in most cases due to telomerase activity) and shortening reactions (generally due to replication and controlled processing) (Jain and Cooper, 2010). Telomerase is capable of adding telomeric repeats to chromosome ends de novo. The enzyme works as a ribonucleoprotein complex, which consists of a catalytic subunit with reverse-transcriptase activity (called TERT), and an RNA serving as the elongation matrix for telomeres (called TR or TERC) (Greider and Blackburn, 1989). While these two core elements are sufficient for telomerase activity in vitro, biochemical analyses have shown that in vivo telomerase resides in a large complex of about 1 MDa (Schnapp et al, 1998). Some additional components of this large multi-subunit holoenzyme complex have been identified. In particular, the core components of box H/ACA small nucleolar ribonucleoprotein particles (snoRNPs), DKC1 (dyskerin), GAR1, NHP2 and NOP10, are part of the active telomerase complex, and are necessary for proper RNP assembly as well as for TERC stability (Mitchell et al, 1999; Wang and Meier, 2004). More recently, the ATPases RUVBL1 and RUVBL2 have been identified as factors essential for holoenzyme assembly (Venteicher et al, 2008), and TCAB1 (WDR79/WRAP53), identified as a DKC1 interaction partner, was shown to be required for proper localization of CAB box containing small Cajal body (CB)-specific RNPs (scaRNPs) to CBs, including TERC, and is part of the active telomerase complex (Tycowski et al, 2009; Venteicher et al, 2009). The presence of major scaRNA processing and trafficking factors in the telomerase complex hints to an important aspect of telomerase cell biology: the orchestrated maturation of telomerase and interaction with telomeres in the CB. Telomere maintenance by telomerase requires that both TERT and TERC are recruited from distinct subnuclear sites to telomeres during S phase (synthesis phase) (Tomlinson et al, 2008). Like other scaRNAs, TERC contains a common CB-specific localization signal and accumulates in CBs (Jády et al, 2004; Zhu et al, 2004), where it is found together with TERT (Tomlinson et al, 2008). In a cell cycle-dependent manner, telomerase-containing CBs are then recruited to telomeres, suggesting that CBs represent an enzymatic hub in which telomere elongation by telomerase takes place (Jády et al, 2006; Tomlinson et al, 2006; Cristofari et al, 2007). This trafficking model is further supported by telomere elongation defects in the absence of TCAB1 or presence of dysfunctional TCAB1, disrupting TERC accumulation in the CB (Venteicher et al, 2009; Zhong et al, 2011). Nevertheless, so far it remains elusive how telomeres are recruited to CBs, how this selective interaction is regulated and what drives the conversion from telomeres in a closed state, in which telomerase has little or no access, to telomeres in an open, accessible state. Telomerase is usually limiting and, under physiological conditions, acts preferentially on short telomeres (Hemann et al, 2001; Britt-Compton et al, 2009), due to a well-established negative feedback loop mediated in cis by TRF1 and POT1, likely by hiding the 3′-overhang, which serves as a template for telomerase (Loayza and de Lange, 2003). Indeed, diminished loading of POT1 or expression of a dominant-negative version lacking DNA-binding activity leads to telomere elongation by telomerase, and in vitro experiments have shown that POT1 is competing with telomerase for its substrate (Loayza and de Lange, 2003; Ye et al, 2004; Kelleher et al, 2005; Lei et al, 2005). However, POT1 also interacts with TPP1, and both proteins together promote telomerase activity in vitro (Latrick and Cech, 2010). Furthermore, TPP1 has been shown to be required for the recruitment of telomerase to its substrate in vitro and to telomeric chromatin in vivo (Xin et al, 2007; Abreu et al, 2010; Tejera et al, 2010; Zaug et al, 2010; Zhong et al, 2012). While TPP1 has been proposed as a telomerase recruiter, it does not completely fit the definition, since it has initially been described as a negative regulator of telomere length in telomerase-positive cells (Liu et al, 2004; Ye et al, 2004), although these results may in part be attributed to secondary effects, such as the lack of POT1 tethering to telomeres in the absence of TPP1. Based on these bivalent results, a ying-yang model for telomerase recruitment and activity control has been proposed for TPP1–POT1 (Xin et al, 2007). Indeed, recently, a specific patch of amino acids on the surface of TPP1, the TEL patch (TPP1 glutamate (E)- and leucine (L)-rich patch), has been identified as crucial for the TPP1 function in telomerase recruitment and regulation (Nandakumar et al, 2012). Analysis of point mutations within the TEL patch demonstrated that the TEL patch is physically and functionally distant from the portion of TPP1 engaged in end protection, separating these two functions (Nandakumar et al, 2012). Regulating the amount of telomeric repeats added by telomerase also involves how long telomerase can act on a given chromosome end. Therefore, turning off telomerase needs to be regulated in addition to the recruitment step and processivity control. In a recent study, Chen et al (2012) described the human CST (CTC1, STN1 and TEN1) complex as a terminator of telomerase activity (Chen et al, 2012). CST competes with POT1–TPP1 for telomeric DNA and is increasingly enriched on telomeric DNA during late S/G2 phase, correlating with the period in the cell cycle when telomerase action is terminated. In agreement with a suppression of telomerase action, depletion of any of the three CST complex members led to a steady increase in telomere length in a telomerase-dependent manner. The authors suggested that CST binds to telomerase-extended 3′-ends and thereby suppresses telomerase access and further elongation (Chen et al, 2012). However, depletion of STN1 has been reported not to affect telomere length in various telomerase-positive cellular contexts (Wang et al, 2012) and murine CTC1-null cells do not exhibit the reported telomere lengthening phenotype (Gu et al, 2012). The proposed model is nonetheless appealing, as human CST subunits stimulate DNA polymerase α-primase (Casteel et al, 2009) and therefore CST binding to telomerase-extended 3′-ends could initiate a switch from telomere elongation to fill-in synthesis. This mechanism could provide an autonomous end point to telomerase action at single telomeres, ensuring that every telomere is extended by telomerase once and only once during every cell cycle (Chen et al, 2012). We reasoned that the identification of novel telomere-binding proteins would be a first step to the identification of additional factors implicated in telomere biology. SILAC-based quantitative mass spectrometry (MS) has been adapted for DNA–protein interactions (Mittler et al, 2009) and has been used by us and others successfully to identify factors binding to particular functional DNA fragments (Markljung et al, 2009; Butter et al, 2010; Bartels et al, 2011; Butter et al, 2012). For our purpose, we applied this approach to telomeric DNA in order to screen for telomere repeat-binding proteins and identified the protein HOT1 (HMBOX1; homeobox telomere-binding protein 1). HOT1 had previously been described as a putative transcriptional repressor based on reporter gene assays (Chen et al, 2006) and had been identified as a telomere-associated protein by the proteomics of isolated chromatin segments (PICh) approach (Déjardin and Kingston, 2009). Here we demonstrate that HOT1 directly binds to telomeric DNA, and characterize this binding in atomic detail by resolving a crystal structure of the HOT1 homeobox domain in a cocrystal with telomeric DNA. In vivo, HOT1 localizes to a subset of telomeres with a higher degree of HOT1–telomere association in cellular contexts of elevated telomere processing. In addition, we show that HOT1 associates with the active telomerase complex and that HOT1 is required for telomerase chromatin binding. These findings suggest that HOT1 contributes to the association of telomerase with telomeres and telomere length maintenance in various cellular settings, and classify HOT1 as the first direct telomere-binding protein that acts as a positive regulator of telomere length. Results Identification of HOT1 as a direct telomere repeat-binding protein To identify telomere-binding proteins we used polymerized biotinylated double-stranded oligonucleotides of the telomeric sequence (5′-TTAGGG-3′) and a scrambled control sequence (5′-GTGAGT-3′), separately immobilized on paramagnetic streptavidin beads and incubated with heavy and light SILAC-labelled nuclear extracts from HeLa cells, respectively. Specific binding of proteins is detected by incubation of cell lysates encoded by ‘heavy’ amino acids (15N- and 13C-labelled Lys and Arg) with the bait sequence, while a control sequence is incubated with ‘light’, nonlabelled amino acids. Specific binders display a differential SILAC ratio, whereas background binders have a 1:1 ratio. After mild washing, bead fractions were combined and captured proteins were analysed by quantitative, high-resolution MS (Cox and Mann, 2008) (Figure 1A). Figure 1.Detection of specific telomere-interacting proteins. (A) A schematic of the quantitative SILAC-based DNA interaction screen with DNA oligonucleotides containing either the telomeric repeat or a control sequence. Specific interaction partners are differentiated from background binders by a SILAC ratio other than 1:1. (B) MS spectra of representative peptides from the ‘forward’ pull-down experiment. The heavy peptide partners are easily detected (red dots), while the light partner is barely observable (blue dots) in the mass spectrum. (C) Two-dimensional interaction plot: known shelterin components cluster together with HOT1, demonstrating enrichment at the telomere sequence compared to the control sequence. (D) Summary of the MS data for HOT1 and the core shelterin components from the SILAC-based DNA–protein interaction screens carried out with nuclear extracts derived from HeLa and murine ES cells. Download figure Download PowerPoint We identified all the six core shelterin components with a SILAC ratio of about 10 or higher in the ‘forward’ and about 0.1 in the ‘reverse’ experiment, in which we had switched the labels (Figure 1B–D, Supplementary Figure S1 and Supplementary Table S1). In contrast, none of the proteins known to interact with shelterin were identified with SILAC ratios sufficiently high to be consistent with telomere binding, demonstrating that this approach was very stringent and exclusively detected telomere repeat-binding proteins and their strong interaction partners (Figure 1C). In addition to the shelterin components, we found the protein HOT1 with a high SILAC ratio that clustered with those of the shelterin components (Figure 1B–D, Supplementary Figure S1 and Supplementary Table S1). This indicates that HOT1 must either strongly associate with the shelterin complex or directly bind to the 5′-TTAGGG-3′ repeats. To verify that the HOT1 identification was not cancer-, cell- or species-specific, we repeated our telomere-binding assay with SILAC-labelled nuclear extracts derived from mouse embryonic stem cells (ES cells). Again, all components of the shelterin complex and HOT1 were identified with SILAC ratios, indicating specific binding to the telomere repeats (Figure 1D and Supplementary Table S2). Here we also identified the two paralogues, POT1a and POT1b, which result from a gene duplication of the Pot1 gene in the rodent lineage (Hockemeyer et al, 2006), underscoring the specificity of our assay for direct telomere-binding proteins. Hence, HOT1 is a putative telomere repeat-binding protein conserved in mammalian cells. HOT1 contains a homeobox domain (Chen et al, 2006), suggesting that it may bind DNA directly. To determine whether HOT1 was detected in our assay due to direct binding to the 5′-TTAGGG-3′ repeats, we performed DNA-binding assays with HOT1 in vitro. Recombinant HOT1 bound specifically to telomeric repeats, whereas no binding to the negative control repeat fragments (5′-GTGAGT-3′) was detected (Figure 2A). Exhibiting similar binding behaviour as TRF1, HOT1 was not enriched on any of the subtelomeric variant repeats 5′-TCAGGG-3′, 5′-TGAGGG-3′ and 5′-TTGGGG-3′, nor on the C. elegans telomere 5′-TTAGGC-3′ repeat sequence (Wicky et al, 1996; Figure 2A). To test whether HOT1 also associates with telomeres in vivo, we performed chromatin immunoprecipitation (ChIP) experiments with extracts from HeLa cells using an antibody directed against HOT1. Similar to TRF2, HOT1 IPs showed enrichment of telomeric DNA in comparison to two negative controls (anti-GFP antibody and IgG; Figure 2B). Thus, HOT1 is a direct and specific telomere repeat-binding protein. Figure 2.The DBD of human HOT1 recognizes telomeric DNA in a sequence-specific manner. (A) Sequence-specific pull-down of recombinant HOT1, TRF1 (positive control) and TBP (TATA-binding protein, negative control). Proteins were incubated with dsDNA of telomeric repeats (5′-TTAGGG-3′), the control sequence (5′-GTGAGT-3′), the subtelomeric repeat variants (5′-TCAGGG-3′, 5′-TGAGGG-3′ and 5′-TTGGGG-3′, as well as the C. elegans telomere repeat 5′-TTAGGC-3′). All DNA substrates were concatemerized from 60 bp oligonucleotides to larger DNA fragments (on average at least 1 kb). (B) ChIP of telomeric DNA using antibodies against HOT1, TRF2 (positive control), GFP and IgG (negative controls). Representative slotblot images are shown for ChIP from HeLa extracts after hybridization with a telomeric and genomic control. Input dilutions demonstrate the linearity of the signals acquired. (C) Structure of the DBD of HOT1 bound to double-stranded telomeric DNA. The protein is shown as a cartoon representation (orange), whereas DNA is shown as a stick model (grey). The interacting amino acid residues in HOT1 are shown as blue sticks, water molecules as red spheres and protein–DNA contacts are visualized as green dashed lines. (D) Schematic representation of all protein–DNA contacts in the complex. (E) Sequence-specific pull-down of FLAG–HOT1 and selected single mutations to investigate binding specificity. Proteins were incubated with either telomeric repeats (5′-TTAGGG-3′) or a control oligonucleotide (5′-GTGAGT-3′). (F) Atomic details of DNA sequence recognition by HOT1. K335 of helix 3 is involved in direct hydrogen bonding to O6 of G8 and O6 of G9. N332 of helix 3 specifically recognizes A11′ of the complementary strand by forming two direct H-bonds with the bicyclic ring system of A11′ (N6 and N7) (left panel). R271 of the N-terminal arm binds two bases of an AT base pair, directly to T9′ and via a water-mediated H-bond to A12 (right panel).Source data for this figure is available on the online supplementary information page. Source Data for Figure 2A [embj2013105-sup-0001-SourceData-S1.pdf] Source Data for Figure 2B [embj2013105-sup-0002-SourceData-S2.pdf] Source Data for Figure 2E [embj2013105-sup-0003-SourceData-S3.pdf] Download figure Download PowerPoint HOT1 recognizes telomeric DNA by means of its homeodomain Driven by these findings and with the aim to fully understand the molecular interactions between HOT1 and telomeric DNA, we crystallized the DNA-binding domain (DBD) of HOT1 with telomeric DNA. In order to identify a construct suitable for crystallization, we initially tested six different HOT1 fragments for their DNA-binding ability (Supplementary Figure S2). The three longer constructs Q144–A345, L156–A345 and G233–A345 all bound to immobilized telomeric dsDNA baits, demonstrating that the homeodomain of HOT1 is sufficient for recognizing telomeric DNA and that integrity of the predicted N-terminal POU-specific (POUs) domain (Chi et al, 2002) is not required. The three shorter constructs (P242–A345, P254–A345 and R271–A345) were not able to bind to the bait DNA (Supplementary Figure S2). For crystallization, we reconstituted and purified telomeric DNA complexes with all three binding constructs, but only one (G233–A345) yielded crystals when reconstituted with a duplex telomeric DNA (5′-cTGTTAGGGTTAGGGTTAG-3′ and 3′-ACAATCCCAATCCCAATCt-5′) similar to the one present in the crystal structures of the TRF1 and TRF2 homeodomains bound to telomeric DNA (Court et al, 2005). The optimized crystals diffracted to 2.9 Å resolution and we could solve the structure by molecular replacement using the NMR model of the human HOT1 homeodomain (residues 268–343, PDB entry 2CUF, unpublished data from RIKEN Structural Genomics/Proteomics Initiative). In the orthorhombic crystals, the DNA forms an infinite double helix via a C–T nonWatson–Crick base pairing of the single-base overhangs (not shown). Two copies of the HOT1 DBD are bound to one duplex DNA that comprises two and a half 5′-TTAGGG-3′ repeats in a regular and undistorted B-form conformation (Supplementary Figure S3). As expected, and in accordance with the NMR model, the homeodomain of HOT1 folds into a small structure of three consecutive helices, α1 (res. 276–288), α2 (res. 293–309) and α3 (res. 322–342), separated by a loop (α1–α2) and a turn (α2–α3; Figures 2C and 3A, and Supplementary Figure S3). The N-terminal residues 233–266 and the two C-terminal residues were not defined by electron density and were, thus, not built. Figure 3.Comparison of the molecular recognition of telomeric DNA by HOT1 and TRFs. (A) Schematic representation of the domain structure of the homeobox domains of TRF1, TRF2 and HOT1. Residues involved in DNA binding are marked with an asterisk (HOT1) or diamond (TRF1). Strictly conserved residues are shown with white font on red background and conserved residues are written in red font. (B) Superposition of structures of the HOT1 DBD and TRF1 DBD bound to telomeric DNA. Both binding domains recognize a different set of DNA bases, resulting in a different positioning relative to the 5′-TTAGGG-3′ motif. Download figure Download PowerPoint As reported for TRF1 and TRF2, each copy of the HOT1 homeodomain binds to the major groove of the DNA double helix around a 5′-TTAGGG-3′ motif (Figure 2C and Supplementary Figure S3). Typical for homeobox domains, binding to DNA is mainly mediated by an N-terminal unstructured arm (267–276), the loop between α1 and α2, and the C-terminal α3 (Figure 2C). Binding and sequence recognition is achieved through a combination of either water-mediated or direct contacts with the phosphate backbone (on both sides of the major groove) and the DNA nucleobases in the major (α3) and minor groove (N-terminal a
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