The Basic Domain of TRF2 Directs Binding to DNA Junctions Irrespective of the Presence of TTAGGG Repeats
2006; Elsevier BV; Volume: 281; Issue: 49 Linguagem: Inglês
10.1074/jbc.m608778200
ISSN1083-351X
AutoresNicole Fouché, Anthony J. Cesare, Smaranda Willcox, Sezgin Özgür, Sarah A. Compton, Jack D. Griffith,
Tópico(s)Chromosomal and Genetic Variations
ResumoThe replication of long tracts of telomeric repeats may require specific factors to avoid fork regression (Fouché, N., Özgür, S., Roy, D., and Griffith, J. (2006) Nucleic Acids Res., in press). Here we show that TRF2 binds to model replication forks and four-way junctions in vitro in a structure-specific but sequence-independent manner. A synthetic peptide encompassing the TRF2 basic domain also binds to DNA four-way junctions, whereas the TRF2 truncation mutant (TRF2ΔB) and a mutant basic domain peptide do not. In the absence of the basic domain, the ability of TRF2 to localize to model telomere ends and facilitate t-loop formation in vitro is diminished. We propose that TRF2 plays a key role during telomere replication in binding chickenfoot intermediates of telomere replication fork regression. Junction-specific binding would also allow TRF2 to stabilize a strand invasion structure that is thought to exist at the strand invasion site of the t-loop. The replication of long tracts of telomeric repeats may require specific factors to avoid fork regression (Fouché, N., Özgür, S., Roy, D., and Griffith, J. (2006) Nucleic Acids Res., in press). Here we show that TRF2 binds to model replication forks and four-way junctions in vitro in a structure-specific but sequence-independent manner. A synthetic peptide encompassing the TRF2 basic domain also binds to DNA four-way junctions, whereas the TRF2 truncation mutant (TRF2ΔB) and a mutant basic domain peptide do not. In the absence of the basic domain, the ability of TRF2 to localize to model telomere ends and facilitate t-loop formation in vitro is diminished. We propose that TRF2 plays a key role during telomere replication in binding chickenfoot intermediates of telomere replication fork regression. Junction-specific binding would also allow TRF2 to stabilize a strand invasion structure that is thought to exist at the strand invasion site of the t-loop. Telomeres are nucleoprotein structures that protect the ends of chromosomes and are essential for regulating the replicative lifespan of somatic cells. The DNA component of the mammalian telomere consists of long double-stranded (ds) 2The abbreviations used are: ds, double-stranded; ss, single-stranded; EM, electron microscopy; TRF2ΔB, TRF2 basic domain truncation mutant; aa, amino acid(s); WRN, Werner syndrome helicase; BLM, Bloom syndrome helicase. 2The abbreviations used are: ds, double-stranded; ss, single-stranded; EM, electron microscopy; TRF2ΔB, TRF2 basic domain truncation mutant; aa, amino acid(s); WRN, Werner syndrome helicase; BLM, Bloom syndrome helicase. tracts of the hexameric repeat unit TTAGGG (2Moyzis R.K. Buckingham J.M. Cram L.S. Dani M. Deaven L.L. Jones M.D. Meyne J. Ratliff R.L. Wu J.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6622-6626Crossref PubMed Scopus (1854) Google Scholar) that ends with a G-rich 3′ single-stranded (ss) overhang (3Henderson E.R. Blackburn E.H. Mol. Cell Biol. 1989; 9: 345-348Crossref PubMed Scopus (258) Google Scholar). Telomeric DNA is thought to be organized into a t-loop “end-capping” structure by the telomere-binding proteins TRF1, TRF2, and POT1 and the proteins that bind to them, TIN2, TPP1, and Rap1 (4Griffith 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 (1904) Google Scholar, 5de Lange T. Genes Dev. 2005; 19: 2100-2110Crossref PubMed Scopus (2240) Google Scholar). This higher order structure may enable cells to distinguish chromosome ends from random double-strand breaks. Large blocks of telomere repeat sequences can be lost when these end-capping proteins are disrupted, or problems are encountered during DNA replication or repair (for review, see Ref. 6Lansdorp P.M. Trends Biochem. Sci. 2005; 30: 388-395Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). This typically results in p53- and Rb-mediated senescence or cellular crisis, as evidenced by end-to-end fusions of chromosomes, ATM-dependent activation of p53, and apoptosis (for review, see Ref. 7de Lange T. Oncogene. 2002; 21: 532-540Crossref PubMed Google Scholar).Much has been learned about the properties of TRF1 and TRF2 including their binding to DNA and the effects of their ablation or overexpression in the cell. We observed that TRF1 forms filamentous structures on long tracts of telomeric DNA in vitro (8Griffith J. Bianchi A. de Lange T. J. Mol. Biol. 1998; 278: 79-88Crossref PubMed Scopus (124) Google Scholar), whereas TRF2 binds preferentially to the telomeric DNA at the junction between the duplex repeats and the ss overhang (9Stansel R.M. de Lange T. Griffith J.D. EMBO J. 2001; 20: 5532-5540Crossref PubMed Scopus (404) Google Scholar). Both TRF1 and TRF2 contain a similar Myb domain at their COOH terminus that mediates their binding to ds telomeric DNA (10Broccoli D. Smogorzewska A. Chong L. de Lange T. Nat. Genet. 1997; 17: 231-235Crossref PubMed Scopus (749) Google Scholar). TRF1 and TRF2 differ in their NH2 termini, however, which are rich in either acidic residues in TRF1 or basic residues in TRF2. The function of the basic domain of TRF2 is poorly understood. Deletion of this domain (TRF2ΔB) does not impede the DNA binding activity of TRF2 or its localization to telomeres in vivo, but expression of TRF2ΔB resulted in stochastic deletions of telomeric DNA, generation of t-loop-sized telomeric circles, cell cycle arrest, and induction of senescence in human cells (11Wang R.C. Smogorzewska A. de Lange T. Cell. 2004; 119: 355-368Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 12Smogorzewska A. van Steensel B. de Lange T. Cell. 1998; 92: 401-413Abstract Full Text Full Text PDF PubMed Scopus (1428) Google Scholar). In addition, recent evidence suggested that the basic domain, but not the Myb domain, was required for TRF2 association with photo-induced double-strand breaks in non-telomeric DNA in human fibroblasts (13Bradshaw P.S. Stavropoulos D.J. Meyn M.S. Nat. Genet. 2005; 37: 193-197Crossref PubMed Scopus (192) Google Scholar).Relatively little is known about the replication of mammalian telomeric DNA in vivo; however, experiments in ciliates and budding yeast have provided insight into how this occurs in other eukaryotes. During each round of replication, all but the very end of the telomere is replicated by the conventional semi conservative polymerase machinery (14Greider C.W. Annu. Rev. Biochem. 1996; 65: 337-365Crossref PubMed Scopus (900) Google Scholar). Leading strand sequences eroded in the last round of replication (end replication problem) can be restored by the reverse transcriptase telomerase (15Greider C.W. Blackburn E.H. Cell. 1987; 51: 887-898Abstract Full Text PDF PubMed Scopus (839) Google Scholar), whereas the lagging strand is concurrently elongated by polymerases α and δ using the newly formed G strand as the template (16Diede S.J. Gottschling D.E. Cell. 1999; 99: 723-733Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar).A possible complication of replication at the telomere is the requirement for protecting the DNA ends from recognition by DNA repair factors while still allowing the DNA to be accessible to the replication machinery. Also, replication of telomeric DNA tends to stall in vitro (17Ohki R. Ishikawa F. Nucleic Acids Res. 2004; 32: 1627-1637Crossref PubMed Scopus (85) Google Scholar), and long blocks of telomeric repeats are highly unstable when transformed into Escherichia coli cells that lack recombination enzymes, suggesting difficulties with DNA replication through the telomeric tract. 3N. Fouché, unpublished data. 3N. Fouché, unpublished data. Furthermore, the G-rich strand of telomeric DNA has the tendency to form G-quartets (18Wang Y. Patel D.J. Structure. 1993; 1: 263-282Abstract Full Text PDF PubMed Scopus (1166) Google Scholar), and the complementary cytosine-rich strand can fold into an intercalated tetramer called the i-motif (19Phan A.T. Gueron M. Leroy J.L. J. Mol. Biol. 2000; 299: 123-144Crossref PubMed Scopus (182) Google Scholar).The fact that human telomeres are replicated as rapidly as the bulk DNA (20Ten Hagen K.G. Gilbert D.M. Willard H.F. Cohen S.N. Mol. Cell. Biol. 1990; 10: 6348-6355Crossref PubMed Scopus (100) Google Scholar) suggests that in addition to the standard replicative machinery, telomere-targeted factors may exist to actively facilitate its rapid replication. Direct evidence for the requirement of such telomeric factors at the replication fork was recently discovered in the fission yeast Schizosaccharomyces pombe, where the telomere-binding protein Taz1 is required for efficient replication of telomeres (21Miller K.M. Rog O. Cooper J.P. Nature. 2006; 440: 824-828Crossref PubMed Scopus (199) Google Scholar). Also, in addition to the normal replicative helicases present at forks, the RecQ helicases WRN and BLM, implicated in premature aging diseases, have been shown to be important for proper telomere replication and maintenance in human cells (22Crabbe L. Verdun R.E. Haggblom C.I. Karlseder J. Science. 2004; 306: 1951-1953Crossref PubMed Scopus (487) Google Scholar, 24Bai Y. Murnane J.P. Hum. Genet. 2003; 113: 337-347Crossref PubMed Scopus (58) Google Scholar).The RecQ helicases have been shown to unwind G quartets (25Yang Q. Zhang R. Wang X.W. Spillare E.A. Linke S.P. Subramanian D. Griffith J.D. Li J.L. Hickson I.D. Shen J.C. Loeb L.A. Mazur S.J. Appella E. Brosh Jr., R.M. Karmakar P. Bohr V.A. Harris C.C. J. Biol. Chem. 2002; 277: 31980-31987Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) and promote branch migration of four-stranded junctions similar to chickenfoot structures (26Constantinou A. Tarsounas M. Karow J.K. Brosh R.M. Bohr V.A. Hickson I.D. West S.C. EMBO Rep. 2000; 1: 80-84Crossref PubMed Scopus (336) Google Scholar, 27Karow J.K. Constantinou A. Li J.L. West S.C. Hickson I.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6504-6508Crossref PubMed Scopus (418) Google Scholar). In human cells TRF2 co-localizes and physically interacts with WRN (28Machwe A. Xiao L. Orren D.K. Oncogene. 2004; 23: 149-156Crossref PubMed Scopus (101) Google Scholar), and it binds to and stimulates the activities of both the WRN and BLM helicases in vitro (29von Opresko P.L. Kobbe C. Laine J.P. Harrigan J. Hickson I.D. Bohr V.A. J. Biol. Chem. 2002; 277: 41110-41119Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). TRF1 and POT1 have also been shown to regulate WRN and BLM unwinding of telomeric substrates in vitro (30Opresko P.L. Mason P.A. Podell E.R. Lei M. Hickson I.D. Cech T.R. Bohr V.A. J. Biol. Chem. 2005; 280: 32069-32080Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 31Opresko P.L. Otterlei M. Graakjaer J. Bruheim P. Dawut L. Kolvraa S. May A. Seidman M.M. Bohr V.A. Mol. Cell. 2004; 14: 763-774Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Furthermore, in vitro overexpression of TRF1 and TRF2 led directly to replication fork stalling (17Ohki R. Ishikawa F. Nucleic Acids Res. 2004; 32: 1627-1637Crossref PubMed Scopus (85) Google Scholar), suggesting that the telomere binding factors also have a direct effect on the replication machinery.We recently discovered a new feature of telomeric DNA that may explain this requirement for the RecQ helicases during telomere replication. Using electron microscopy (EM) and model replication fork templates that mimic a replication fork that had transited a long block of telomeric repeats, we discovered that telomeric DNA is inherently more slippery than non-repeat-containing DNA, such that the replication forks are able to more easily transition back and forth between non-regressed and fully regressed states (1Fouché N. Özgür S. Roy D. Griffith J. Nucleic Acids Res. 2006; (in press)PubMed Google Scholar). During replication, repeat-containing DNA could, therefore, spend a significantly larger fraction of time in the partially regressed state, characterized by a Holliday junction or “chickenfoot” structure, than other DNAs. We believe that this presents a significant problem to the cell, where these four-stranded structures could result in recruitment of unwanted recombination factors or lead to deleterious recombination events if repaired. p53 will also bind to stalled chickenfoot structures with great affinity, suggesting that it may have the ability to halt excessive fork regression (32Subramanian D. Griffith J.D. J. Biol. Chem. 2005; 280: 42568-42572Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). These observations led us to ask whether one or both of the primary ds telomere-binding proteins TRF1 and TRF2 might also show some unusual binding with regard to three- and four-way DNA junctions when they occur within telomeric tracts.To test this hypothesis we generated a set of DNA templates including telomeric and non-telomeric replication forks, Holliday junctions, and model telomeres containing 3′ overhangs. Using EM and polyacrylamide gel-shift assays, we evaluated binding to these templates by TRF1, TRF2, and TRF2ΔB as well as a peptide encompassing the basic domain of TRF2 and another similar “mutant” peptide containing a rearrangement of four amino acids (aa).In this paper we show that TRF2, but not TRF1 or TRF2ΔB, is able to target the junctions of replication forks, chickenfoot structures, and Holliday junctions. Junction binding occurred irrespective of the presence of TTAGGG repeats, and a bias for four-stranded junctions was detected. The peptide mimicking the basic domain of TRF2 recapitulated this four-way junction binding, whereas the mutant peptide could not. Furthermore, in the absence of other telomere-binding proteins, TRF2 lacking the basic domain had a reduced ability to target the end of the large model telomeres and facilitate t-loop formation in vitro. We, therefore, suggest a novel role for the previously uncharacterized basic domain of TRF2, which is to facilitate TRF2 binding to chickenfoot intermediates of telomere replication fork regression, presumably preventing their recognition by Holliday junction resolvases. The data are also the first direct demonstration of TRF2 binding to DNA junctions, irrespective of the presence of telomeric repeats.EXPERIMENTAL PROCEDURESDNA Probes and Templates—[γ-32P]ATP end-labeled J12 four-way junction probes (33Lee S. Cavallo L. Griffith J. J. Biol. Chem. 1997; 272: 7532-7539Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), large Holliday-junction DNA templates (HJ575 (34Alani E. Lee S. Kane M.F. Griffith J. Kolodner R.D. J. Mol. Biol. 1997; 265: 289-301Crossref PubMed Scopus (86) Google Scholar)), model non-telomeric replication forks (32Subramanian D. Griffith J.D. J. Biol. Chem. 2005; 280: 42568-42572Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), and model telomere DNA (9Stansel R.M. de Lange T. Griffith J.D. EMBO J. 2001; 20: 5532-5540Crossref PubMed Scopus (404) Google Scholar) were synthesized as previously described. A telomeric probe with a 14-nucleotide overhang was prepared by annealing the [γ-32P]ATP end-labeled oligonucleotide (oligo) 5′-CTAACCCTAACCCTGTCCTAGCAATGTAATCGTCTATGAGTCTG-3′ to the oligo 5′-CAGACTCATAGACGATTACATTGCTAGGACAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3′. A hairpin probe consisting of a 7-nucleotide linker and a 21-bp stem was prepared by heating and stepwise cooling the [γ-32P]ATP end-labeled oligo 5′-CTTATTCACAGACCACGACTCAAAAAAAGAGTCGTGGTCTGTGAATAAG-3′. All annealed products were purified on 10% nondenaturing polyacrylamide gels.Telomeric replication forks were created using a variant of pRST5 (9Stansel R.M. de Lange T. Griffith J.D. EMBO J. 2001; 20: 5532-5540Crossref PubMed Scopus (404) Google Scholar) containing a nicking site directly adjacent to the telomeric repeat tract (1Fouché N. Özgür S. Roy D. Griffith J. Nucleic Acids Res. 2006; (in press)PubMed Google Scholar). The plasmid was nicked with N.BbvC IA (New England Biolabs, Ipswich, MA) and then incubated with the Klenow fragment (exo–) of DNA polymerase 1 (New England Biolabs) and 0.5 mm each of dTTP, dATP, and dGTP to generate an ss tail by strand displacement of the repeat tract. The ss tail was converted to a ds tail by annealing a 228-fold molar excess of the oligo 5′-CCCTAACCCTAACCCTAACCCTAA-3′ to the template for 30 min at 37 °C in 100 mm NaCl and ligating with T4 DNA ligase (400 units, New England Biolabs) at 16 °C overnight in 50 mm NaCl, 10 mm Tris-HCl (pH 7.9), and 1 mm dithiothreitol. When required, the replication templates were linearized with XmnI (New England Biolabs) for 1 h at 37°C.Proteins and Peptides—NH2-terminal His6-tagged human TRF1, TRF2, and TRF2ΔB were purified to homogeneity from baculovirus-infected insect cells by the method of Bianchi et al. (35Bianchi A. Smith S. Chong L. Elias P. de Lange T. EMBO J. 1997; 16: 1785-1794Crossref PubMed Scopus (268) Google Scholar) except that a Talon™ metal affinity resin (Clontech, Palo Alto, CA) was employed instead of nickel-nitrilotriacetic acid (35Bianchi A. Smith S. Chong L. Elias P. de Lange T. EMBO J. 1997; 16: 1785-1794Crossref PubMed Scopus (268) Google Scholar). p53 as well as the carboxyl-terminal domain of the p53 protein comprising amino acid residues 311–393 were purified as previously described (36Wu L. Bayle J.H. Elenbaas B. Pavletich N.P. Levine A.J. Mol. Cell. Biol. 1995; 15: 497-504Crossref PubMed Scopus (112) Google Scholar).Two peptides, each containing an NH2-terminal biotin motif, were synthesized by the UNC Micro-Protein Facility, University of North Carolina School of Medicine, Chapel Hill, NC (Fig. 1B). The first peptide (Peptide_B) consisted of aa 2–31 of wild type human TRF2 (10Broccoli D. Smogorzewska A. Chong L. de Lange T. Nat. Genet. 1997; 17: 231-235Crossref PubMed Scopus (749) Google Scholar), whereas the second peptide (Peptide_M) comprised a 4-aa sequence rearrangement of the first peptide.Electron Microscopy—Binding assays of TRF2 and TRF2ΔB to the model telomere were done as previously described (9Stansel R.M. de Lange T. Griffith J.D. EMBO J. 2001; 20: 5532-5540Crossref PubMed Scopus (404) Google Scholar). Complexes of p53, TRF1, TRF2, TRF2ΔB, and both peptides with Holliday junction or replication fork DNA were formed by incubating a 25:1 molar ratio of protein monomer:DNA in a 20-μl volume of EM buffer (20 mm HEPES (pH 7.5), 0.1 mm EDTA, 0.5 mm dithiothreitol, 75 mm KCl) for 20 min at room temperature. A 5× molar excess of streptavidin (Molecular Probes, Eugene, OR) to peptide was added to samples containing biotin-tagged peptide for 5 min at room temperature. Samples were fixed with 0.6% (w/v) glutaraldehyde for 5 min at room temperature followed by filtration through 2-ml columns of 2% agarose beads (50–150 μm, Agarose Bead Technologies, Tampa, FL) pre-equilibrated with 0.01 m Tris-HCl (pH 7.6), 0.1 mm EDTA. The purified samples were prepared for EM by rotary shadow-casting with tungsten as previously described (37Griffith J.D. Christiansen G. Annu. Rev. Biophys. Bioeng. 1978; 7: 19-35Crossref PubMed Scopus (181) Google Scholar). An FEI Tecnai 12 electron microscope equipped with a Gatan ultrascan camera (model US4000SP) were used to photograph images.Mobility Shift Assays—Reaction mixtures (10 μl) containing probes (10 nm) and the proteins (see legends to Figs. 3 and 5 for details) were incubated at room temperature for 20 min in EM buffer. The mixtures were adjusted to 10% glycerol and loaded on 3.5% non-denaturing polyacrylamide gels in 45 mm Tris borate, 1 mm EDTA. The gels were run at 140 V for 1 h at 4°C, dried, analyzed by autoradiography, and quantified using a Storm 840 PhosphorImager (GE Healthcare).FIGURE 3TRF2 junction binding is biased toward four-way junctions. A, binding of p53, TRF2, and TRF1 to the telomeric replication fork templates (tel. RF) and the non-telomeric replication fork templates (non-tel. RF) was visualized by EM and quantified. Percentages are calculated as a fraction of all replication forks counted (three-way and four-way junctions combined). Only molecules with protein bound at the junction of the replication fork or chickenfoot structure were considered junction-bound molecules. Data are represented as the mean ± S.D. B, Holliday junction (HJ) templates bound by TRF1 or TRF2 were similarly quantified. C, percentages of TRF2 and p53 binding to replication forks are calculated as a fraction of molecules with the same shape. D, mobility shift assay of the γ-32P-labeled J12 junction probe alone (lane 1) or bound by p53 (at a molar ratio of 30:1 protein:probe, lane 2), TRF2 (25:1, lane 3; 50:1, lane 4), an 82-aa COOH-terminal fragment of p53 containing the basic domain (81:1, lane 5), and TRF1 (50:1, lane 6; 101:1, lane 7).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5The basic domain of TRF2 is required for binding to four-way junctions. A and B, mobility shift assays of the γ-32P-labeled J12 junction probe alone (lanes 1) or bound by the basic peptide (A, lanes 2–9), the mutant peptide (A, lanes 9–15), streptavidin alone (A, lane 16); TRF2 (B, lanes 2–8), and TRF2ΔB (B, lanes 9–14). In samples containing peptide, the molar ratio of streptavidin protein:peptide used was 5:1. C, apparent dissociation constants (Kd) for TRF2, TRF1, TRF2ΔB, the basic peptide (Pep_B), and the mutant peptide (Pep_M) binding to the small γ-32P end-labeled telomere probe or to the γ-32P end-labeled Holliday junction probe (J12). The last lane (Adjusted Kd) shows the apparent Kd values for Holliday junction binding adjusted by the specific binding factors obtained via EM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Calculating Kd—GraphPad Prism (GraphPad Software, Inc., San Diego, CA) was used for nonlinear regression of the data obtained from the mobility shift assays. The one-site hyperbolic binding equation used was % probe shifted = (Bmax × [nm protein])/([nm protein] + Kd). In experiments with the Holliday junction probe, dissociation constant (Kd) values were converted to association constant (Ka) values (Ka = 1/Kd) then multiplied by the EM-specific binding factor (see “Results”) (Ka,apparent = Ka × % EM junction binding) and again converted to a dissociation constant (Kd,apparent = 1/Ka,apparent (nm)).RESULTSTo examine the binding of TRF1 and TRF2 to replication forks and Holliday junctions and to compare this with previous studies of p53 (32Subramanian D. Griffith J.D. J. Biol. Chem. 2005; 280: 42568-42572Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), a series of large model templates were constructed for EM. Previously we described the generation of a model replication fork consisting of an ∼500-bp arm extended from a 3-kilobase linear or circular DNA at a unique site, which is at the end of a 500-bp cassette consisting of a random but G-less sequence (32Subramanian D. Griffith J.D. J. Biol. Chem. 2005; 280: 42568-42572Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). We constructed a new replication fork template based on this design from a plasmid containing a 560-bp telomeric cassette (Fig. 1A). In this DNA two arms of the Y-fork molecule contain telomere repeats leading up to the fork junction. In addition, a Holliday junction template containing 500-bp arms of plasmid-derived DNA extended from the well known J12 junction was prepared as previously described (33Lee S. Cavallo L. Griffith J. J. Biol. Chem. 1997; 272: 7532-7539Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar).Purified TRF1 and TRF2 were incubated with the template DNAs and prepared for EM. EM binding experiments were conducted in parallel with purified p53. For these studies p53 was chosen for comparison with TRF2 not in an effort to argue for the biological relevance if its binding to replication forks or Holliday junctions but, rather, that it is of similar size to TRF2, it binds these junctions tightly without altering their structure significantly, and we have on hand data of p53 binding to the very DNA templates used in this study. Examination of fields of molecules from incubations of TRF2 with the model telomere replication fork revealed several DNA-protein configurations. When a 25:1 molar ratio of TRF2 monomers to DNA template was used, approximately one-third of all molecules contained a particle of TRF2 bound at the center of the stalled fork junction (Fig. 2A). Of these molecules, a subset of the forks had regressed, generating chickenfoot structures, and TRF2 was also observed bound at these junctions (inset, Fig. 2A). The remainder of the DNA consisted of DNA templates with no protein bound (the majority), a fewer number of DNAs containing TRF2 protein bound elsewhere on the DNA template but not at the fork junction, and more frequently, aggregates of two or more DNAs bound by a large mass of TRF2 (data not shown). These aggregates became more abundant in binding preparations containing molar ratios of TRF2 monomers to DNA template greater than 25:1. Of the TRF2-bound molecules where TRF2 was not located at the fork junction, the majority had TRF2 bound sufficiently close to the fork that we presumed it was bound along the TTAGGG tracts. Quantification of the results from scoring hundreds of molecules is described below. Although the TRF2 particles varied in size, in most cases the size range was much less variable when bound to the center of the three- or four-way junction than in the instances when TRF2 was observed bound along the duplex TTAGGG tracts (Fig. 2A, inset), and the particle size was suggestive of TRF2 dimers or tetramers. When parallel experiments were performed with TRF1, the telomeric repeats were complexed by multiple protein particles (Fig. 2B) resembling the TRF1 filaments formed along duplex TTAGGG tracts as previously described (8Griffith J. Bianchi A. de Lange T. J. Mol. Biol. 1998; 278: 79-88Crossref PubMed Scopus (124) Google Scholar).FIGURE 2TRF2 binds DNA junctions in vitro. A and C, discrete complexes of TRF2 bound to the junctions of the three-stranded replication forks and the four-stranded chickenfoot structures (arrow, inset) (A, telomeric template; C, non-telomeric template). B, extensive, filamentous binding of TRF1 to a model telomeric replication forks. D, TRF2 bound to the center of Holliday junction template DNAs. The bar is equivalent to 450 bp in the panels showing full-length molecules.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For comparison, TRF1 and TRF2 were incubated with the random sequence replication fork template lacking TTAGGG repeats. The binding of TRF1 to this template was low (quantified in Fig. 3A), as expected from the lack of telomeric repeats. Of great interest, however, was the observation that incubation of TRF2 with this template led to discrete TRF2 complexes at the three- or four-way junctions (Fig. 2C). In molecules in which the forks had regressed into four-way chickenfoot forms, TRF2 complexes were more frequently observed bound to the DNA and almost always at the four-way junction (inset, Fig. 2C and quantified below). This led us to examine TRF2 binding to Holliday junction DNA containing 500-bp arms (Fig. 2D). Indeed, by EM we found that TRF2 bound well to these structures, localizing to the center of the four-way junction. By comparison, TRF1 bound less well to these structures, and when it did, the binding appeared to be random (quantified in Fig. 3B).We previously examined the binding of p53 to random sequence replication fork templates (32Subramanian D. Griffith J.D. J. Biol. Chem. 2005; 280: 42568-42572Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) as well as to large Holliday junction templates (33Lee S. Cavallo L. Griffith J. J. Biol. Chem. 1997; 272: 7532-7539Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). We were, thus, able to combine these results in a comparison of TRF1 and TRF2 binding to these templates (Fig. 3, A–C). An average of 179 molecules over three experiments was counted per experimental condition.Incubation of p53 (25:1) with the telomeric replication fork template resulted in observation of p53 at the fork and the frequency (69 ± 3 versus 8 ± 1% elsewhere on the DNA; Fig. 3A) was comparable with previous observations with non-telomeric forks (56% at the fork versus 15% bound elsewhere (32Subramanian D. Griffith J.D. J. Biol. Chem. 2005; 280: 42568-42572Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar)). We could not assess TRF1 binding to telomeric replication fork templates because of its extensive binding to the telomeric tract. Also, because the number of DNA molecules contained within the TRF2 aggregates could not be determined by EM, they were not included in the total number of DNAs counted. Thus, the actual level of TRF2 binding to the telomeric replication fork is likely to be higher than the value cited here (34 ± 21 versus 6 ± 2% elsewhere on the DNA; Fig. 3A) because the values were calculated as the percentage of the individual (non-aggregated) DNAs only. The relatively wide spread in calculated TRF2 binding is most likely due to a variation in the abundance of TRF2 aggregates in these samples, because when TRF2 binding in these samples was evaluated as a percentage of individually bound molecules, the S.D. was small (76 ± 5% bound at the junction versus 24 ±
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