2021 journal article

Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA

JOURNAL OF BIOLOGICAL CHEMISTRY, 297(3).

By: H. Pan n, P. Kaur n, R. Barnes *, A. Detwiler *, S. Sanford *, M. Liu  n, P. Xu n, C. Mahn n ...

MeSH headings : Cell Adhesion Molecules / metabolism; Cell Adhesion Molecules / physiology; DNA / metabolism; DNA-Binding Proteins / metabolism; Humans; Microscopy, Atomic Force / methods; Models, Molecular; Multiprotein Complexes / metabolism; Protein Binding; Protein Isoforms / metabolism; Shelterin Complex / metabolism; Shelterin Complex / physiology; Telomere / metabolism; Telomere-Binding Proteins / metabolism; Telomere-Binding Proteins / physiology; Telomeric Repeat Binding Protein 1 / metabolism; Telomeric Repeat Binding Protein 1 / physiology; Telomeric Repeat Binding Protein 2 / metabolism; Telomeric Repeat Binding Protein 2 / physiology
TL;DR: Analysis of DNA molecular structures promoted by TRF1-TIN2 interaction supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation. (via Semantic Scholar)
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Added: October 26, 2021

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation. TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation. Telomeres are nucleoprotein structures that prevent the degradation or fusion of the ends of linear chromosomes, which are threatened by at least seven distinct DNA damage response (DDR) pathways (1Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1344) Google Scholar, 2Muraki K. Nyhan K. Han L. Murnane J.P. Mechanisms of telomere loss and their consequences for chromosome instability.Front. Oncol. 2012; 2: 135Crossref PubMed Google Scholar, 3de Lange T. Shelterin-mediated telomere protection.Annu. Rev. Genet. 2018; 52: 223-247Crossref PubMed Scopus (280) Google Scholar). Human telomeres contain ~2–20 kb of TTAGGG repeats and a G-rich 3′ overhang of ~50–400 nt in length (1Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1344) Google Scholar, 4Wright W.E. Tesmer V.M. Huffman K.E. Levene S.D. Shay J.W. Normal human chromosomes have long G-rich telomeric overhangs at one end.Genes Dev. 1997; 11: 2801-2809Crossref PubMed Scopus (572) Google Scholar). In humans, a specialized six-protein shelterin complex consisting of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1 binds specifically to the unique sequence and structure at telomeres to protect chromosome ends. Prevention of telomeres from being falsely recognized as double-strand DNA breaks and regulation of DNA repair protein access depend on the biochemical activities of shelterin proteins and their collaborative actions with other proteins involved in the genome maintenance pathways (5Cech T.R. Beginning to understand the end of the chromosome.Cell. 2004; 116: 273-279Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 6Songyang Z. Liu D. Inside the mammalian telomere interactome: Regulation and regulatory activities of telomeres.Crit. Rev. Eukaryot. Gene Expr. 2006; 16: 103-118Crossref PubMed Scopus (38) Google Scholar, 7Verdun R.E. Karlseder J. Replication and protection of telomeres.Nature. 2007; 447: 924-931Crossref PubMed Scopus (370) Google Scholar, 8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. 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Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice.Nature. 2011; 469: 102-106Crossref PubMed Scopus (556) Google Scholar). The main protein–protein and protein–DNA interactions at telomeres have been investigated using crystallography, biochemical assays, yeast two-hybrid systems, coimmunoprecipitation, as well as visualization of shelterin subcomponents in vitro and in vivo using fluorescence imaging (3de Lange T. Shelterin-mediated telomere protection.Annu. Rev. Genet. 2018; 52: 223-247Crossref PubMed Scopus (280) Google Scholar). Among shelterin components, both TRF1 and TRF2 specifically recognize double-stranded telomeric DNA through the Myb/SANT domain facilitated by homodimerization through the TRFH domain (12Broccoli D. Chong L. Oelmann S. Fernald A.A. Marziliano N. van Steensel B. Kipling D. Le Beau M.M. de Lange T. Comparison of the human and mouse genes encoding the telomeric protein, TRF1: Chromosomal localization, expression and conserved protein domains.Hum. Mol. Genet. 1997; 6: 69-76Crossref PubMed Scopus (80) Google Scholar, 13Broccoli D. Smogorzewska A. Chong L. de Lange T. Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2.Nat. Genet. 1997; 17: 231-235Crossref PubMed Scopus (741) Google Scholar). However, TRF1 and TRF2 display distinct DNA-binding properties and functions. TRF1 and TRF2 contain an acidic and a basic domain, respectively, at their N-termini (14Poulet A. Pisano S. Faivre-Moskalenko C. Pei B. Tauran Y. Haftek-Terreau Z. Brunet F. Le Bihan Y.V. Ledu M.H. Montel F. Hugo N. Amiard S. Argoul F. Chaboud A. Gilson E. et al.The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA.Nucleic Acids Res. 2012; 40: 2566-2576Crossref PubMed Scopus (55) Google Scholar). 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Jaune E. Foucher K. Cherfils-Vicini J. Aeby E. et al.TRF2-Mediated control of telomere DNA topology as a mechanism for chromosome-end protection.Mol. Cell. 2016; 61: 274-286Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 18Van Ly D. Low R.R.J. Frolich S. Bartolec T.K. Kafer G.R. Pickett H.A. Gaus K. Cesare A.J. Telomere loop dynamics in chromosome end protection.Mol. Cell. 2018; 71: 510-525.e516Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In comparison, TRF1 represses telomere fragility by preventing DNA replication fork stalling at telomeres (19Sfeir A. Kosiyatrakul S.T. Hockemeyer D. MacRae S.L. Karlseder J. Schildkraut C.L. de Lange T. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication.Cell. 2009; 138: 90-103Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar) and promotes parallel pairing of telomeric DNA tracts (20Griffith J. Bianchi A. de Lange T. TRF1 promotes parallel pairing of telomeric tracts in vitro.J. Mol. Biol. 1998; 278: 79-88Crossref PubMed Scopus (123) Google Scholar, 21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (44) Google Scholar). A flexible domain in TRF1 enables the two Myb domains in the TRF1 dimer to interact with DNA independently and to mediate looping of telomeric DNA (22Bianchi A. Stansel R.M. Fairall L. Griffith J.D. Rhodes D. de Lange T. TRF1 binds a bipartite telomeric site with extreme spatial flexibility.EMBO J. 1999; 18: 5735-5744Crossref PubMed Scopus (163) Google Scholar). TIN2 itself does not have binding affinity to either double-stranded or single-stranded DNA (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar). However, it is a core shelterin component that bridges double-stranded (TRF1 and TRF2) and single-stranded telomeric DNA-binding proteins (TPP1-POT1) (24Kim S.H. Beausejour C. Davalos A.R. Kaminker P. Heo S.J. Campisi J. TIN2 mediates functions of TRF2 at human telomeres.J. Biol. Chem. 2004; 279: 43799-43804Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 25Houghtaling B.R. Cuttonaro L. Chang W. Smith S. A dynamic molecular link between the telomere length regulator TRF1 and the chromosome end protector TRF2.Curr. Biol. 2004; 14: 1621-1631Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 26Kim S.H. Davalos A.R. Heo S.J. Rodier F. Zou Y. Beausejour C. Kaminker P. Yannone S.M. Campisi J. Telomere dysfunction and cell survival: Roles for distinct TIN2-containing complexes.J. 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A shared docking motif in TRF1 and TRF2 used for differential recruitment of telomeric proteins.Science. 2008; 319: 1092-1096Crossref PubMed Scopus (187) Google Scholar). TIN2 stabilizes both TRF1 and TRF2 at telomeres (31Kim S.H. Han S. You Y.H. Chen D.J. Campisi J. The human telomere-associated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro.EMBO Rep. 2003; 4: 685-691Crossref PubMed Scopus (42) Google Scholar, 32Ye J.Z. Donigian J.R. van Overbeek M. Loayza D. Luo Y. Krutchinsky A.N. Chait B.T. de Lange T. TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres.J. Biol. Chem. 2004; 279: 47264-47271Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The loss of the TRF1 or TRF2-binding domains in TIN2 triggers a DNA damage response (33Takai K.K. Kibe T. Donigian J.R. Frescas D. de Lange T. Telomere protection by TPP1/POT1 requires tethering to TIN2.Mol. Cell. 2011; 44: 647-659Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Binding of TPP1 to TIN2 is required for POT1-mediated telomere protection (34Frescas D. de Lange T. Binding of TPP1 protein to TIN2 protein is required for POT1a,b protein-mediated telomere protection.J. Biol. Chem. 2014; 289: 24180-24187Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). As an integral component of the "TIN2/TPP1/POT1 processivity complex," TIN2 functions together with TPP1/POT1 to stimulate telomerase processivity (35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar). Furthermore, TIN2 directly interacts with the cohesin subunit SA1 and plays a key role in a distinct SA1-TRF1-TIN2-mediated sister telomere cohesion pathway that is largely independent of the cohesin ring subunits (8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. Protein requirements for sister telomere association in human cells.EMBO J. 2007; 26: 4867-4878Crossref PubMed Scopus (80) Google Scholar, 36Canudas S. Smith S. Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells.J. Cell Biol. 2009; 187: 165-173Crossref PubMed Scopus (122) Google Scholar). Binding of TRF1-TIN2 to telomeres is regulated by the poly(ADP-ribose) polymerase Tankyrase 1 (37Smith S. Giriat I. Schmitt A. de Lange T. Tankyrase, a poly(ADP-ribose) polymerase at human telomeres.Science. 1998; 282: 1484-1487Crossref PubMed Scopus (891) Google Scholar). ADP-ribosylation of TRF1 by Tankyrase 1 reduces its binding to telomeric DNA in vitro, and the depletion of Tankyrase 1 using siRNA leads to mitotic arrest and persistent telomere cohesion that can be rescued by depletion of TIN2 (8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. Protein requirements for sister telomere association in human cells.EMBO J. 2007; 26: 4867-4878Crossref PubMed Scopus (80) Google Scholar, 36Canudas S. Smith S. Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells.J. Cell Biol. 2009; 187: 165-173Crossref PubMed Scopus (122) Google Scholar, 38Dynek J.N. Smith S. Resolution of sister telomere association is required for progression through mitosis.Science. 2004; 304: 97-100Crossref PubMed Scopus (216) Google Scholar). Three distinct TIN2 isoforms have been identified in human cell lines (35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar, 39Kaminker P.G. Kim S.H. Desprez P.Y. Campisi J. A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.Cell Cycle. 2009; 8: 931-939Crossref PubMed Scopus (35) Google Scholar, 40Smith S. The long and short of it: A new isoform of TIN2 in the nuclear matrix.Cell Cycle. 2009; 8: 797-798Crossref PubMed Scopus (2) Google Scholar) that include TIN2S (354 AAs), TIN2L (451 AAs), and TIN2M (TIN2 medium, 420 AAs). TIN2S, TIN2L, and TIN2M share the same TRF1, TRF2, and TPP1-binding domains and localize to telomeres (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar, 35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar, 39Kaminker P.G. Kim S.H. Desprez P.Y. Campisi J. A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.Cell Cycle. 2009; 8: 931-939Crossref PubMed Scopus (35) Google Scholar). Consistent with its key role in telomere maintenance, germline inactivation of TIN2 in mice is embryonic lethal (41Chiang Y.J. Kim S.H. Tessarollo L. Campisi J. Hodes R.J. Telomere-associated protein TIN2 is essential for early embryonic development through a telomerase-independent pathway.Mol. Cell. Biol. 2004; 24: 6631-6634Crossref PubMed Scopus (61) Google Scholar). Removal of TIN2 leads to the formation of telomere dysfunction-induced foci (TIFs). Importantly, clinical studies further highlight the biological significance of TIN2 in telomere protection (42Savage S.A. Giri N. Baerlocher G.M. Orr N. Lansdorp P.M. Alter B.P. TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita.Am. J. Hum. 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However, since TIN2 itself does not directly bind to DNA and instead serves as a "mediator/enhancer" for shelterin and telomerase activities, defining TIN2's distinct function at the molecular level has been challenging. The bottleneck for studying TIN2 lies in the fact that results from bulk biochemical assays do not fully reveal the heterogeneity and dynamics of the protein–protein and protein–DNA interactions. Furthermore, cell-based assays only provide information on the outcomes from downstream effectors after the knocking down of TIN2 that also removes TRF1 and TRF2 from telomeres. These approaches do not allow us to investigate the molecular structures and dynamics in which TIN2 directly participates. In vivo, the amount of TIN2 is sufficient for binding every TRF1 and TRF2 molecule (44Takai K.K. Hooper S. Blackwood S. Gandhi R. de Lange T. In vivo stoichiometry of shelterin components.J. Biol. Chem. 2010; 285: 1457-1467Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), while TPP1 and POT1 are ~10-fold less than TRF1 and TIN2. Thus, it is important to study the DNA-binding properties of TRF1-TIN2 complexes. To fill this important knowledge gap, we applied complementary single-molecule imaging platforms, including atomic force microscopy (AFM) (45Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29: 175-187Crossref PubMed Scopus (88) Google Scholar, 46Wang H. Nora G.J. Ghodke H. Opresko P.L. Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding.J. Biol. Chem. 2011; 286: 7479-7489Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 47Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. Riehn R. Opresko P.L. Wang H. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 6: 20513Crossref PubMed Scopus (20) Google Scholar), total internal reflection fluorescence microscopy (TIRFM) (48Erie D.A. Weninger K.R. Single molecule studies of DNA mismatch repair.DNA Repair. 2014; 20: 71-81Crossref PubMed Scopus (46) Google Scholar), and the DNA tightrope assay to monitor TRF1-TIN2-mediated DNA compaction and DNA-DNA bridging (49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar, 50Countryman P. Fan Y. Gorthi A. Pan H. Strickland J. Kaur P. Wang X. Lin J. Lei X. White C. You C. Wirth N. Tessmer I. Piehler J. Riehn R. et al.Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates.J. Biol. Chem. 2018; 293: 1054-1069Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 51Pan H. Jin M. Ghadiyaram A. Kaur P. Miller H.E. Ta H.M. Liu M. Fan Y. Mahn C. Gorthi A. You C. Piehler J. Riehn R. Bishop A.J.R. Tao Y.J. et al.Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA.Nucleic Acids Res. 2020; 48: 5639-5655Crossref PubMed Google Scholar). Through using DNA substrates on different length scales (6 and 270 TAAGGG repeats), these imaging platforms provide complementary results demonstrating that both TIN2S and TIN2L facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. In some cases, TRF1-TIN2 is capable of mediating the bridging of multiple copies of telomeric DNA fragments. Importantly, our results demonstrate that TIN2 protects the disassembly of TRF1-TIN2-mediated DNA-DNA bridging by Tankyrase 1. In addition, the N-terminal domain of TPP1 inhibits TRF1-TIN2-mediated DNA-DNA bridging. In summary, this study uncovered the unique biophysical function of TIN2 as a telomeric architectural protein, acting together with TRF1 to mediate interactions between distant telomeric sequences. Tankyrase 1 and TPP1 regulate TRF1-TIN2-mediated DNA-DNA bridging. Furthermore, this work establishes a unique combination of single-molecule imaging platforms for future examination of TIN2 disease variants and provides a new direction for investigating molecular mechanisms underlying diverse TIN2 functions. A previous study suggested that TIN2 modulates the bridging of telomeric DNA by TRF1 (31Kim S.H. Han S. You Y.H. Chen D.J. Campisi J. The human telomere-associated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro.EMBO Rep. 2003; 4: 685-691Crossref PubMed Scopus (42) Google Scholar). However, the bulk biochemical assays using short telomeric DNA (six telomeric repeats) did not provide information regarding the structure and dynamics of the TRF1-TIN2-DNA complex. To investigate the molecular function of TIN2, we applied AFM imaging to investigate how TIN2 affects the telomeric DNA-DNA pairing mediated by TRF1 at the single-molecule level on longer telomeric DNA substrates (270 TTAGGG repeats). We purified TRF1 (Fig. S1A) and obtained TIN2S (1–354 amino acids, 39.4 kDa) and TIN2L (1–451 amino acids, 50.0 kDa) proteins purified from insect cells (Fig. 1A and Fig. S1D). Previously, we established an AFM imaging-based calibration method to investigate the oligomeric states and protein–protein interactions by correlating AFM volumes of proteins and their molecular weights (45Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29: 175-187Crossref PubMed Scopus (88) Google Scholar, 47Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. Riehn R. Opresko P.L. Wang H. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 6: 20513Crossref PubMed Scopus (20) Google Scholar, 52Wang H. Yang Y. Erie D.A. Characterization of protein-protein interactions using atomic force microscopy.in: Schuck P. Protein Interactions Biophysical approaches for the Study of Complex Reversible Systems. Springer Science+Business Media, LLC, Berlin, Germany2007: 39-78Crossref Google Scholar). AFM volumes of TRF1 alone in solution showed two distinct peaks, which were consistent with TRF1 monomers (51 KDa) and dimers (102 KDa, Fig. S1B). In addition, based on the population of TRF1 under the monomer and dimer peaks (53Wang H. DellaVecchia M.J. Skorvaga M. Croteau D.L. Erie D.A. Van Houten B. UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity.J. Biol. Chem. 2006; 281: 15227-15237Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), the estimated TRF1 dimer equilibrium dissociation constant (Kd) is 18.4 nM (Fig. S1C). Meanwhile, AFM volumes of purified TIN2S at 41.3 nm3 (±28.3 nm3) and TIN2L at 41.9 nm3 (±12.8 nm3) were consistent with the notion that TIN2 does not interact with itself (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), and TIN2 exists in a monomeric state in solution (Fig. S1D). Furthermore, we conducted size-exclusive chromatography using TRF1 and TIN2S and confirmed the presence of TRF1 dimers, TIN2 monomers, as well as the interaction between TRF1 and TIN2S in solution (Fig. S2). To further validate the activities of TIN2, we used electrophoresis mobility shift assays (EMSAs) to verify the interaction of TIN2 with TRF1 on a double-stranded telomeric DNA substrate (48 bp containing three TTAGGG repeats, Fig. S3, A–C). Consistent with previous studies (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), EMSA experiments showed that TIN2S and TIN2L did not directly bind to telomeric dsDNA (Fig. S3A). Both TRF1-TIN2S and TRF1-TIN2L induced a clear supershift of the telomeric DNA substrate compared with TRF1 alone (Complex III in Fig. S3, B and C), indicating the formation of stable TRF1-TIN2-telomeric DNA complexes. Next, to study TRF1-TIN2 DNA binding at the single-molecule level, we used the linear DNA substrate (5.4 kb) that contains 1.6 kb (270 TTAGGG) telomeric repeats in the middle region that is 35%–50% from DNA ends (T270 DNA, Experimental procedures, Fig. 1A) (21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (44) Google Scholar, 49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar). 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Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar). To study the function of TIN2, we preincubated TRF1 without or with TIN2 (either TIN2S or TIN2L), followed by the addition