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Genes to Cells (2004) 9, 205-218. doi:10.1111/j.1356-9597.2004.00719.x
© 2004 Blackwell Publishing or its licensors
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Molecular mechanisms of DNA end-loop formation by TRF2

Shige H. Yoshimura1,*, Hugo Maruyama1, Fuyuki Ishikawa2,3, Rieko Ohki3,a and Kunio Takeyasu1

1 Department of Responses to Environmental Signals and Stresses, and 2 Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kitashirawkawa-oiwake-cho Sakyo-ku Kyoto, 606-8502, Japan
3 Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
In the telomere region of human chromosomes, the (TTAGGG)n sequence stretches over several kilobases and forms a distinct higher-order structure with various proteins. Telomere repeat binding factors (TRFs) bind specifically to this sequence and play critical roles in the maintenance of telomere structure and function. Here, we prepared a series of linear DNA carrying a stretch of telomeric sequence ((TTAGGG)n, ~1.8 (kb) with different end-structures and observed their higher-order complexes with TRFs by atomic force microscopy. TRF2 molecules exclusively bound to the telomeric DNA region at several different places simultaneously mainly as a dimer, and often mediated DNA loop formation by forming a tetramer at the root. These multiple-binding, multimerization and DNA loop formation by TRF2 were observed regardless of the DNA-end structure (blunt, 3'-overhanging, telomeric, non-telomeric). However, when the DNA end carried the telomeric-3'-overhanging region, the loop was frequently formed at the end of the DNA. Namely, the TRF2-mediated DNA loop formation is independent of the end-structure and the 3'-overhanging TTAGGG sequence is responsible for the stabilization of the loop. TRF1 also bound to the telomeric DNA as a dimer, but did not mediate DNA loop formation by itself. These results provide a new insight into the molecular mechanism of DNA end-loop formation by TRFs.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
A special DNA-protein complex at the telomere region distinguishes the end of chromosomes from other random DNA breaks and protects this region from chemical and enzymatic attacks associated with DNA repair and recombination. In human chromosomes, telomeric DNA is composed of a long repetitive sequence of TTAGGG (de Lang et al. 1990; Moyzis et al. 1988). Several telomere-specific proteins carrying a Myb-like DNA binding motif have been identified in yeasts (Cooper et al. 1997; Konig et al. 1996; Spink et al. 2000), nematodes (Kim et al. 2003), plants (Chen et al. 2001; Hwang et al. 2001; Yang et al. 2003; Yu et al. 2000) and vertebrates (Bilaud et al. 1996; Chong et al. 1995; Konrad et al. 1999; Smilenov et al. 1998). Telomere repeat binding factors (TRF1 and TRF2) are specifically localized to the telomere region of human chromosomes and are involved in the maintenance of telomere length (Smogorzewska et al. 2000), and the protection of telomeres from end-to-end fusions (van Steensel et al. 1998). TRF1 and TRF2 are homologous proteins and exclusively bind to the (TTAGGG)n sequence via the Myb-related DNA binding domain (Bilaud et al. 1997; Broccoli et al. 1997).

The structures of the TRF proteins themselves and in complex with DNA have been partially resolved by X-ray crystallography (Fairall et al. 2001) and NMR (Nishikawa et al. 2001). The single Myb domain in the TRF1 protein specifically recognizes the AGGGTT sequence and binds to DNA as a dimer (Bianchi et al. 1997, 1999; Nishikawa et al. 2001). The DNA binding domain at the C-terminus of TRF1 and the dimerization domain, which is located in the N-terminal half of the protein, are connected by a flexible hinge, thus this protein binds to DNA with spatial flexibility (Bianchi et al. 1999). Although the structure of TRF2 complexed with DNA has not been resolved yet, it may be similar to TRF1 since the Myb-like DNA binding domains in these proteins are well conserved at an amino acid level. In the N-terminal domain, however, there is a prominent sequence difference; TRF1 has a region rich in acidic residues, whereas TRF2 has a basic domain at its N-terminus. However, it remains to be determined whether this lack of sequence homology in this region gives rise to functional differences.

The molecular mechanisms underlying chromosome protection by TRF proteins have been studied by electron microscopy. It has been observed that the telomeric DNA end forms a large DNA loop (t-loop) (Griffith et al. 1999; Munoz-Jordan et al. 2001; Murti & Prescott 1999). In the human t-loop model, TRF2 preferentially binds to the border of the double-stranded telomeric DNA and mediate a DNA end-loop formation (Griffith et al. 1999; Stansel et al. 2001). At the branching point of the loop, the 3'-end of the single-stranded TTAGGG sequence invades into the double stranded DNA region of the same molecule (d-loop). This t-loop/d-loop model could explain how TRF2 protects DNA ends from the enzymatic attack and end-to-end fusion. However, the molecular mechanism of such t-loop/d-loop formation is speculative, especially the processes that involve TRF2. In this report, we utilized an in vitro system to elucidate the molecular mechanisms of DNA loop formation by TRF2. Under well-defined conditions, the loop was formed first in the double-stranded region of the telomeric DNA by DNA-dependent multimerization of TRF2 independent of the 3'-overhanging, and then the loop was preferentially stabilized at the border of the double-stranded and the single stranded regions. The 3'-overhanging plays a role in the stabilization of the loop at this border possibly by forming a specific structure, such as a quadruplex. In contrast to the previous human model, our results suggest that the ‘d-loop’ formation would require additional factors.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Preparation of 3'-overhanging telomeric DNA

We constructed an in vitro system that can be used for structural analysis of the human telomere (Rieko Ohki & Fuyuki Ishikawa, unpublished observation). A plasmid containing ~1.8 kb of TTAGGG repeats (pT2AG3, ~4.8 kb, Fig. 1A) was stably maintained in a bacterial strain, STBL2, to minimize the probability of DNA recombination within the repetitive sequence and to maintain the full-length telomeric DNA insert. The purified plasmid was linearized by a restriction enzyme, either BbsI or SspI. BbsI cleaves the plasmid at the very edge of the TTAGGG repeats (Fig. 1A), producing a 5'-overhang of TAAC (Fig. 1B). SspI cleaves the plasmid at a single site, which is not located within the TTAGGG repeat region, and produces blunt ends (Fig. 1C). Since the telomere region of human chromosome carries a 3'-overhanging single-stranded region of several hundred base pairs (Henderson & Blackburn 1989; Wright et al. 1997), the 3'-overhanging region was enzymatically introduced to the end of the linearized plasmids by {lambda}-exonuclease. Since {lambda}-exonuclease has a low activity towards 5'-overhangs, the BbsI-digested plasmid was treated with DNA polymerase I Klenow fragment to fill in the 5'-overhanging region prior to the {lambda}-exonuclease treatment (designated as ‘telomeric blunt’ in Fig. 1B). After implementing this protocol, the plasmid carried a 3'-overhanging of (TTAGGG)n at one end (designated as ‘telomeric 3'-overhanging’ in Fig. 1B) and a non-telomeric 3'-overhang at another end. The plasmid treated with SspI and {lambda}-exonuclease carried non-telomeric 3'-overhangs at both ends (designated as ‘non-telomeric 3'-overhanging’ in Fig. 1C).



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  		Figure 1  DNA and proteins used in this study. (A) A schematic illustration of pT2AG3. A plasmid used in this study (pT2AG3) has a 1.8 kb repeating insert of the human telomeric sequence TTAGGG. The recognition site of the restriction enzyme BbsI is located at the very end of the telomeric sequence, while the SspI site is located at ~200 bp from the end. (B) A telomeric DNA end was produced by digestion of the plasmid with BbsI followed by filling the end using Klenow fragment (designated as ‘telomeric blunt’) and by further treating with {lambda}-exonuclease to produce 3'-overhanging (designated as ‘telomeric 3'-overhanging’). (C) A non-telomeric DNA end was produced by digestion of the plasmid with SspI (designated as ‘non-telomeric blunt’) followed by treatment with {lambda}-exonuclease (designated as ‘non-telomeric 3'-overhanging’). (D) TRF proteins used in this study. Hexahistidine-tagged TRF1 and TRF2 were purified and analysed by SDS-PAGE and immunoblotting. The proteins were detected either by CBB staining (left panel) or using anti-TRF antibodies (right panels). (E) Electrophoretic mobility shift assay. Purified (His)6-tagged TRF1 or TRF2 was incubated with 32P-labelled double-stranded telomeric DNA ((TTAGGG)28). A 20-fold unlabelled DNA was added as a competitor.

 
End-structure of 3'-overhanging telomeric DNA

The end structures of these DNA molecules were observed by atomic force microscopy (AFM). The linearized and blunt-ended plasmid DNAs had a typical shape of double-stranded DNA edge at both ends of the strand (‘rigid end’) (Fig. 2A, left column). The contour length of the plasmid was ~1600 nm (Fig. 2A, bottom left panel), which well matches to the theoretical length of a 4.8 kb duplex DNA strand (1630 nm). There were a small number of DNA molecules shorter than 1600 nm. This was probably due to the shortening of the repetitive DNA in bacteria induced by recombination.



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  		Figure 2  AFM images of linearized plasmid carrying telomeric DNA. The pT2AG3 was treated as described in Figure 1 and observed by AFM. (A) A summary of AFM images. The structure of the linearlized plasmid was depicted in top panels. Two representative AFM images were shown in middle panels. The contour length of double-stranded DNA region was measured on AFM images and plotted in bottom panels. After {lambda}-exonuclease treatment, the double-stranded region was reduced by ~130 nm (corresponding to ~380 bp). The ‘rigid’ (open triangle), ‘branched’ (filled triangle), ‘globular’ (asterisk) structures are indicated in AFM images in (A) and summarized in (B) together with ‘stretched’ form. The frequencies of each form in various types of DNA are shown below in the absence or presence of 100 mM K+. Bars, 100 nm.

 
On the contrary, the end structures of 3'-overhanging DNAs were variable (Fig. 2A, middle and right columns). The averaged length of the double-stranded region of these plasmids was ~1470 nm (Fig. 2A, bottom panels), indicating that the length of the single-stranded region at each DNA end was approximately 190 bp. Figure 2(B) summarizes the end structures observed in these plasmids. A significant population (~44%) had several different kinds of structures unique to single-stranded DNA, including a self-annealed form with several small branches (branched), a single small globular form (globular) and a stretched form (stretched).

The small globular form was observed only at one end of the plasmid carrying telomeric overhanging DNA, but not observed in other types of DNA (Fig. 2B) including the plasmid without the telomeric DNA insert (data not shown), indicating that this structure is specific to the 3'-overhanging telomeric DNA end. Since it was previously reported that single-stranded DNA of the (TTAGGG)n sequence could form a quadruplex structure (Parkinson et al. 2002), we tested the possibility that the small globular structure observed here is the quadruplex structure and obtained the following results; (i) This structure was never observed in other types of DNA end than telomeric 3'-overhang. (ii) The size of the globular structure (~3.9 nm in width) was almost the same as that of the G-quadruplex resolved by X-ray crystallography (3.8 nm). (iii) When K+ was depleted from the solution, the globular structure was not observed (Fig. 2B) (K+ is necessary for the G-quadruplex structure) (Parkinson et al. 2002). (iv) When the DNA was heated and then gradually cooled down to induce the quadruplex structure, the frequency of globular structure formation increased to 20%. Thus, all these results suggest that the small globular structure observed at the telomeric overhanging DNA end is a quadruplex or closely related structure specific to the single-stranded telomeric sequence.

TRF2 binds to the telomeric DNA and mediates DNA loop formation

TRF2 has a high affinity for the duplex form of (TTAGGG)n DNA (Bilaud et al. 1996; Broccoli et al. 1997). The hexahistidine-tagged TRF2 protein was expressed by baculovirus expression system and purified by affinity chromatography (Fig. 1D). The high affinity of this tagged protein to the TTAGGG repeat was confirmed both by gel-shift analysis (Fig. 1E) and DNA pull-down assay using telomeric double-stranded DNA (data not shown).

The purified TRF2 was incubated with DNA carrying telomeric 3'-overhanging end (Fig. 3A) and observed by AFM. Approximately 53% of DNA molecules had bound proteins. Among those, three different kinds of DNA/protein structures were observed (type I, type II and type III, Fig. 3B,E,H, respectively). The type I complex (33% of the total DNA, n = 315) had TRF2 protein molecules bound to DNA, but a DNA loop was not formed (Fig. 3B). The position of the bound protein on the DNA was determined by measuring the distance from the closer end of the DNA strand. Statistical analysis of the binding site revealed that TRF2 always bound within one third from an end of the DNA strand (i.e. ~550 nm from one end) (Fig. 3C). Since TRF2 did not bind to the plasmid DNA without telomeric DNA insert (less than 1%, n = 141, data not shown), it can be concluded that TRF2 exclusively bound to telomeric DNA, confirming the result from gel shift assay (Fig. 1E). The number of proteins bound to a single DNA molecule varied between 1 and 8, with an average of 2.9 (Fig. 3D), demonstrating that multiple TRF2 molecules simultaneously bind to the telomeric DNA at several different places.



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  		Figure 3  TRF2 mediates DNA loop formation within the telomeric sequence. The structure of the linearized plasmid carrying telomeric 3'-overhanging is depicted in (A). When TRF2 was incubated with this DNA, three different types of interaction were detected; TRF2 bound to the telomeric DNA without forming a loop (type I, B), or with mediating a DNA loop in the middle (type II, E) or at the end (type III, H) of the telomeric sequence. The frequency of type I, II and III observations were 33%, 15% and 5%, respectively (total 315 plasmids were analysed). Proteins involved in (open triangles) and not involved in (filled triangles) loop formation are indicated in the AFM images. The distances between the bound protein and the closer end of the DNA chain in the type I complex were measured and summarized in (C). The number of protein bindings in a single type I DNA was quantified and plotted in (D). In the type II complex, the position (F) and the length (G) of the DNA loop were analysed. For the type III complex, the contour length of the loop was measured (I). The angles between the loop forming DNAs at the loop junction in the type III complex are summarized in (J). Bars, 100 nm.

 
In the type II complexes (15% of total DNA), TRF2 bound to DNA and mediated DNA loop formation in the middle of the DNA strand (Fig. 3E). The loop was always formed within the telomeric DNA region (Fig. 3F,G). The type III complexes (5% of total DNA) also had a DNA loop, but in this case it was located at the end of the DNA chain (Fig. 3H). In the type III complexes, the loop was also always formed within the telomeric DNA region (Fig. 3I).

The angle between the two loop-forming DNA strands at the branching point of the type III complex was always less than 90 degrees (Fig. 3J) (see Discussion for detail). Monovalent cations (K+, Na+) did not affect the binding characteristics of TRF2 (data not shown).

Multimerization of TRF2 upon DNA-loop formation

The molecular size of TRF2 was quantified and analysed statistically (Fig. 4). When TRF2 was observed in the absence of plasmid DNA, the size of TRF2 molecules could be divided into two different populations (Fig. 4A). The population with the smaller molecular volume (93% of the total) corresponded to the monomer and that with a larger volume (7% of the total) to the dimer (see Experimental procedures), demonstrating that most of the TRF2 protein existed in monomeric form. TRF2 molecules bound to DNA without forming a loop (type I) were also classified into monomeric and dimeric populations. However, in this case, the dimer occurred more frequently (62% of the total) (Fig. 4B), indicating that TRF2 preferred to form a dimer when it bound to telomeric DNA. Furthermore, the proteins at the branching point of the DNA loop were larger than a dimer; the majority being of a size indicative of a tetramer (Fig. 4C). Notably, this phenomenon did not depend on the position of the loop since tetramerized TRF2 proteins were found at the branching points of both type II (Fig. 4C) and type III complexes (Fig. 4D).



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  		Figure 4  Multimerization of TRF2 on DNA. The size of the TRF2 protein in the AFM images was measured and the molecular volume was calculated after subtracting the tip effect (see Experimental procedures). Representative AFM images (left panels) and quantification of protein volume (right panels) in each type of complex are shown. Most of the TRF2 protein existed as a monomer, without the plasmid DNA (A), whereas a significant population existed as a dimer in the type I complex (B). In type II and III complexes, TRF2 bound to DNA at the branching point of the loop tended to form tetramers, whereas bound proteins not involved in loop formation (C, D) were mainly dimeric. The molecular volumes of the TRF2 monomer, dimer and tetramer were estimated on the basis of molecular weight (see Experimental procedures). Open triangles, filled triangles and arrows in the AFM images and the histograms indicate monomer, dimer and tetramer, respectively.

 
The 3'-overhanging region of telomeric DNA is necessary for loop formation at the DNA end

When the plasmid with the blunt-ended telomeric sequence (‘telomeric blunt’ in Fig. 1B) was incubated with TRF2, similar DNA binding properties were observed (Fig. 5A). Type I complexes were found in 40% of total DNA (n = 179) (Fig. 5A, left panel). The size of the protein corresponded to either the monomer or the dimer (data not shown) as was the case for the overhanging telomeric DNA. Type II complexes were also observed in 9% of total DNA (Fig. 5A, right panel), but type III complexes did not occur. This indicates that the preferred location of the loop at the chain end depends on the existence of the 3'-overhanging region, although TRF2-mediated DNA loop formation per se does not require the 3'-overhanging region of the telomeric sequence.



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  		Figure 5  Loop formation at a DNA end requires the 3'-overhanging region of telomeric DNA. The linearlized plasmid carrying telomeric blunt end (A) or non-telomeric 3'-overhanging ends (B) was incubated with TRF2 and the reaction product was analysed by AFM. DNA with bound protein in the telomeric sequence without (type I, left panels) or with (type II, right panels) a DNA loop was observed. However, an end-loop (type III) was never observed for either form of DNA. The frequency of the occurrence of each structure is indicated in the images (total 179 and 145 plasmids were analysed for (A) and (B), respectively). Bars, 100 nm.

 
To examine whether the end-loop formation depends on the nucleotide sequence of the 3'-overhanging region, TRF2 was incubated with DNA carrying non-telomeric 3'-overhanging ends (‘non-telomeric 3'-overhanging’ in Fig. 1C) and observed by AFM (Fig. 5B). In this case, type I (40% of the total DNA, n = 145) and type II complexes (16% of the total DNA) were detected (Fig. 5B), but the end-loop (type III) was never observed. These results demonstrated that the 3'-overhanging region of the telomeric sequence is necessary for TRF2-mediated end-loop formation.

TRF1 binds to telomeric repeats but does not mediate DNA loop formation

TRF1 is a homologue of TRF2 and, similar to TRF2, binds with high specificity to the (TTAGGG)n duplex DNA (Broccoli et al. 1997). Hexahistidine-tagged TRF1 protein was affinity-purified and analysed by SDS-PAGE followed by immunoblotting (Fig. 1D). As in the case of TRF2, the affinity of the purified TRF1 to telomeric DNA was confirmed by gel mobility-shift assay (Fig. 1E). When incubated with DNA carrying telomeric 3'-overhanging region, TRF1 showed similar DNA-binding characteristics to TRF2, i.e. it specifically bound to the telomeric DNA region within the plasmid (68% of the total DNA, n = 160; Fig. 6A). Multiple bindings of TRF1 on a single DNA molecule were frequently observed and statistical analysis of protein size revealed that TRF1 bound to DNA mainly as a dimer (Fig. 6A, bottom panel), which agrees well with previous reports (Bianchi et al. 1997, 1999; Nishikawa et al. 2001). However, the TRF1 protein did not mediate any DNA loop formations as neither type II (middle-loop) nor type III (end-loop) complexes were formed on telomeric DNA by TRF1.



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  		Figure 6  TRF1 binds to telomeric DNA but does not mediate DNA loop formation. (A) TRF1 binding to telomeric DNA. The linearized plasmid carrying the telomeric 3'-overhanging end was incubated with TRF1. An representative AFM image was shown. Multiple bindings of TRF1 were observed. Sixty-eight percent of DNA molecules were type I complexes. Neither type II nor type III were observed. The contour length of the TRF1 binding site from the closer end of the DNA chain was measured and summarized (middle panel). The volume of bound TRF1 was calculated based on the AFM images as described (Experimental procedures) and plotted as in Figure 4 (bottom panel). Monomers and dimers are indicated with open and filled triangles, respectively. (B) Simultaneous binding of TRF1 and TRF2 to telomeric DNA. Equal amounts of TRF1 and TRF2 were incubated with telomeric 3'-overhanging DNA and observed by AFM. Type I (71% of the total DNA, n = 133), type II (10%) and type III (4%) complexes were observed. The volume of the bound proteins in the type I complex (bottom left) and at the branching point of the type II and the type III complexes (bottom right) were calculated and plotted, as in Figure 4. Monomers (open triangle), dimers (filled triangle) and tetramers (arrow) are indicated in the AFM images and histograms. A protein complex larger than a tetramer was found at the branching point of the type II and III complexes (indicated by an asterisk). (C) Summary of the TRF2-binding assay. The frequencies of unbound DNA and type I, II and III complexes are summarized from the experiments described in Figs 3, 5 and 6. Only when the DNA carrying the telomeric 3'-overhanging was incubated with TRF2, an end-loop (type III) was observed. Bars: 100 nm.

 
When TRF1 and TRF2 were simultaneously incubated with 3'-overhanging telomeric DNA, type I, II, and III complexes were observed (Fig. 6B). Within the type I complexes (71% of the total DNA, n = 133), multiple molecules were observed to bind telomeric DNA as is the case of each of the TRF protein. However, statistical analysis of the protein size revealed that, in addition to monomers and dimers, tetramers were also found within the type I complexes (Fig. 6B, bottom left panel). The frequency of tetramer formation was approximately 28% of the total bound proteins. Since TRF1 and TRF2 proteins by themselves independently bind to DNA as a homo-dimer but not as a homo-tetramer within the type I complexes (Figs 4B and 6A), it is possible that this tetramer is composed of both TRF1 and TRF2 proteins (see Discussion). In type II and type III complexes, protein species larger than tetramers were observed at the branching point of the DNA loop (Fig. 6B, asterisk, bottom right panel). Considering that TRF2, but not TRF1, mediates DNA loop formation by forming a tetramer, TRF1 proteins might be incorporated into the TRF2 complex at the branching point or be located in close proximity to the complex.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
By utilizing telomeric DNA with various end-structures and a procedure which facilitates one-molecule imaging (AFM), we elucidated the structure of the telomeric end of DNA and the mechanisms of DNA loop formation by TRF2. The result also suggested that there is a certain cooperative interaction between TRF1 and TRF2 in end-loop formation. The results of the in vitro binding assay are summarized in Fig. 6C.

Single-stranded DNA with TTAGGG repeats forms a distinct higher-order structure similar to the G-quadruplex structure

Prominent characteristics of telomeric DNA include the repetitive sequences (TTAGGG for human, TTTTGGGG in Oxytricha nova, TG1-3 in budding yeast) and a long-3'-overhang of the G-rich strand (Henderson & Blackburn 1989; McElligott & Wellinger 1997; Wright et al. 1997). Various structures of such G-rich single-stranded DNA have been proposed. A G-quartet model was proposed for telomeric DNA of Oxytricha nova ((TTTTGGGG)n) on the basis of X-ray crystallographic studies (Horvath & Schultz 2001; Kang et al. 1992; Smith & Feigon 1992; Williamson et al. 1989). For human telomeric DNA, a parallel quadruplex structure was recently resolved by X-ray analysis, using a 22mer oligonucleotide containing the telomeric sequence (Parkinson et al. 2002). In this structure, four strands of the GGG sequence are paralleled to form a quadruplex and the intervening TTA sequences are looped out from the quadruplex core. The globular structure at the end of telomeric overhanging DNA observed in the current study had similar characteristics to this G-quadruplex structure (Fig. 2B). The globular structure can be observed only when the DNA carries a telomeric 3'-overhanging, and the size of the blob is similar to that of the G-quadruplex structure resolved by X-ray crystallography. Considering the fact that a single-stranded DNA, but not RNA, does not usually form a blob (Sakai et al. 2003), we propose that this globular structure is a quadruplex or closely related structure specific to the single-stranded telomeric sequence. The functional significance of the quadruplex structure is still an open question but might be involved in the stabilization of DNA end-loop structure.

DNA-dependent multimerization of TFR2 and loop formation

Our results showed that multiple bindings of TRF2 (varying from 1 to 8 per DNA molecule) were quite common within the telomeric DNA (Fig. 3). The analysis of the protein sizes in the AFM images showed that TRF2 binds to the telomeric DNA mainly as a dimer, which is consistent with the previous results (Bianchi et al. 1997). However, monomer binding was also observed in our experimental condition (Fig. 4B). The previous work utilizing gel mobility shift assays reported that TRF1 existed as a dimer and monomer binding was never observed (Bianchi et al. 1997). This is likely to be due to the differences in the protein concentration; an extremely low concentration of the protein was used in the present AFM assay. The ratio of TRF molecule to the binding site (TTAGGG sequence) was approximately 0.3 in the present study, which was several hundreds times lower than those in the gel-shift assays. It might be the case that two independent TRF2 monomers encounter each other during the random sliding along telomeric DNA and form a stable dimer, although this hypothesis needs to be tested in the future.

TRF2 frequently mediated DNA loop formation within telomeric DNA (Fig. 3E,H), and this depended neither on the location of the telomeric DNA within the plasmid (at the end or in the middle of the linearlized DNA) nor on the structure of the DNA ends (blunt-end or 3'-overhanging single-stranded end) (Fig. 5). It should be noted that at least four TRF2 molecules are assembled at the branching point of the loop (Fig. 4C). This indicates that the DNA loop formation was not simply mediated by the dimerization of TRF2, and further suggests that there must be a certain mechanism for inducing the tetramerization of TRF2. This type of DNA-dependent protein multimerization has been observed in other DNA binding proteins including SMC (Structural Maintenance of Chromosome). For example, when bound to DNA, condensin SMC heterodimers undergo further multimerization via a DNA-dependent interaction, resulting in the aggregation of a DNA-protein complex (Yoshimura et al. 2002).

The results obtained in this study show a clear contrast to the previous model, in which a DNA end-loop but not a middle-loop is supposed to be exclusively formed (Griffith et al. 1999). The model has proposed that TRF2 first binds to the border of the double-stranded and the single stranded regions of the telomeric DNA and then mediates a DNA loop formation at the end of the DNA (Stansel et al. 2001). However, our results demonstrated that the middle-loops with varying sizes were formed with a high frequency (15%) regardless of the existence of the 3'-overhanging (Fig. 3E,H). The formation of the middle-loop by TRF2 may be an initial step for t-loop formation. The second step would be the end-structure-dependent stabilization of the loop at the border of double-stranded and single stranded regions.

Tetramerization of TRF2 is probably mediated via N-terminal half of the protein which contains a dimerization domain and a basic domain. It was previously reported that dimerization of TRF2 produces a new interface which is highly reactive with other proteins (Fairall et al. 2001). Upon DNA binding, the TRF2 dimer might undergo a conformational change so that this reactive interface could mediate the tetramerization event. It should be noted that TRF1 by itself did not mediate DNA loop formation, though it specifically bound to telomeric DNA (Fig. 6A). Both TRF1 and TRF2 carry a well-conserved Myb-like DNA binding domain within their C-terminals, which results in high specificity for binding to the (TTAGGG)n sequence. However, the dimerization domains of TRF1 and TRF2 are not well conserved at the amino acid level (Fairall et al. 2001). Furthermore, the N-terminal region of TRF2 is rich in basic residues, whereas the corresponding domain of TRF1 contains more acidic residues. Thus, it can be speculated that basic characteristics of the N-terminal region, as well as the dimerization domain, might be important for the DNA-dependent tetramerization.

The 3'-overhanging region of telomeric DNA clamps the TRF2-mediated DNA loop at the chain end

When linearized DNA with a telomeric 3'-overhanging was used, the frequency of DNA loop formation at DNA ends increased (Fig. 3H). This result indicates that the stability of the DNA end-loop and the structure of the DNA end are closely related. The 3'-overhanging telomeric DNA, but neither the blunt-ended telomeric DNA nor the 3'-overhanging non-telomeric DNA, is required for the stabilization of the DNA loop at the end (Fig. 5).

From the above data, a model of TRF2 function can be deduced (Fig. 7). Multiple TRF2 dimers are formed on telomeric DNA. Two TRF2 dimers on the same DNA molecule occasionally assemble to form a tetramer and mediate DNA loop formation. The TRF2 tetramer then keeps a random sliding on DNA, while keeping the loop structure. When the TRF2 tetramer comes to the end of the double-stranded region, where TRF2 has relatively higher affinity (Stansel et al. 2001), the sliding is stalled and the DNA loop is stabilized there. The quadruplex structure might be involved in this stabilization of the end-loop.



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  		Figure 7  Functional model of TRF2 in end-loop formation. Several TRF2 dimers simultaneously bind to telomeric DNA. Two dimers are associated to form a tetramer, producing a DNA loop within the telomeric DNA. The TRF2 tetramer could then slide along the DNA, and may be stabilized when it reaches the end of the duplex DNA and encounters single-stranded DNA. The quadruplex structure of the single-stranded (TTAGGG)n sequence most likely contributes to this stabilization.

 
It was previously speculated that TRF2 induces a d-loop formation at the branching point of the t-loop, which requires a partial denaturation of double strand DNA (Greider 1999; Griffith et al. 1999). Our results suggest that TRF2 per se cannot induce such a d-loop formation. Since the 3'-overhanging region of the TTAGGG repeat forms a higher-order structure such as G-quadruplex, the denaturation of double-stranded DNA and the invasion of single-stranded DNA would require an energy input. However, the end-loop formation by TRF2 does not require ATP or other energy sources. Furthermore, the angle between the two loop-forming DNA strands at the branching point was always smaller than 90 degrees (Fig. 3J). If the d-loop is formed at the branching point, the angle is likely to be larger than 90 degrees. From the consideration described above, we expect that the formation of the t-loop/d-loop structure in vivo may require other protein components in addition to TRF1 and TRF2.

TRF1 and TRF2 cooperate on telomeric DNA

When both TRF1 and TRF2 were simultaneously incubated with telomeric DNA, a tetrameric protein complex was occasionally formed on telomeric DNA without forming a DNA loop (Fig. 6B). Since neither TRF1 nor TRF2 by itself binds to the telomeric DNA as a tetramer in the absence of a DNA loop (Figs 4B and 6A), this tetrameric complex is likely to contain both TRF1 and TRF2 dimers. At present, our AFM resolution does not allow us to distinguish the individual dimers in the complex due to their similar molecular weights (TRF1: 50 kD, TRF2: 55 kD). Therefore, the actual tetrameric organization within the complex requires further investigation.

Yeast two hybrid assays (Broccoli et al. 1997), structural modelling (Fairall et al. 2001) and immunoprecipitation (Zhu et al. 2000) have all shown that there is no direct interaction between TRF1 and TRF2 monomers. However, our result implies that there may be a certain cooperative association between these proteins when they bind to telomeric DNA, although it is unclear whether TRF1 and TRF2 directly interact with each other. Alternatively, both proteins may simply be located in close proximity to one another, which cannot be resolved using our current AFM technology. Even if this were the case, it appears that TRF1 and TRF2 dimers may cooperate each other in telomeric loop formation. In the presence of TRF1 and TRF2, type II and type III complexes carried a large protein complex (larger than a tetramer) at the branching point of the loop (Fig. 6B). It should be noted that the associated bindings of TRF1 and TRF2 to telomeric DNA did not inhibit DNA loop formation (Fig. 6B,C). Although the functional relationship between TRF1 and TRF2 dimers in DNA loop formation is unclear, this large protein complex probably contains both TRF2 tetramer and TRF1 dimer(s). The elucidation of the higher-order structures mediated by TRF1, TRF2 and other telomeric proteins merits further investigations.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Plasmid and proteins

The plasmid pT2AG3, which contains approximately 1.8 kb of (TTAGGG)n, was constructed as previously described (R Ohki & F. Ishikawa, unpublished observation). Briefly, double-stranded oligo-nucleotide (5'-CTAGATCTTTAGATATCGTCTTCGACGT-3'/5'-CGAAGACGATATCTAAAGATCTAGACGT-3') was inserted into an AatII site of pSVO11 (Tsurimoto & Stillman 1991). Another double-stranded oligonucleotide (5'-AGCTTCAGTGCAGCATATGTCAGA-3'/5'-GATCCAGTCTGACATATGCTGCACTGA-3') was subcloned into pBluescript (Novagen) with BamHI and HindIII sites (pBS-BsgI). The TTAGGG repeat DNA was amplified by PCR as described (Ijdo et al. 1991) and subcloned into pT7Blue(R) (Novagen) (pT7Blue-T2AG3). The T2AG3 fragment was then swapped into pBS-BsgI with NdeI and BamHI sites. The T2AG3 fragment was again cut out with BsgI and BamHI and, after treatment with T4 DNA polymerase to remove 3'-overhanging nucleotides at the BsgI site, subcloned into pSVO11-EcoRV digested with EcoRV and BglII sites.

The plasmid pT2AG3 was amplified in the bacterial strain, STBL2 (Invitrogen). After transformation, several colonies were picked and cultured in LB medium at 30 °C, and the total length of the plasmid was examined with a small-scale DNA preparation. A single clone, carrying the full-length telomeric DNA fragment, was selected and amplified again to obtain a large-scale DNA preparation. The plasmid DNA was digested with either BbsI or SspI (New England BioLabs). The BbsI-digested plasmid was subsequently treated with Klenow fragment (New England BioLabs) to fill in the 5'-overhanging region. The blunt-ended DNA was incubated with {lambda}-exonuclease in a reaction buffer containing 67 mM glycine-KOH (pH 9.4) and 2.5 mM MgCl2 at 16 °C for 3 min

The expression and purification of the TRF1 and TRF2 proteins tagged with hexahistidine have been previously described (R. Ohki & F. Ishikawa, submitted). Briefly, cDNAs encoding N-terminally [His]6-tagged human TRF1 and TRF2 were cloned into pFASTBAC HTa vector (Gibco BRL). Recombinant baculovirus expressing TRF1 and TRF2 were produced by following the manufacturer's protocol. Sf9 cells producing TRF1 and TRF2 were harvested 60 h after infection of virus. The cells were suspended in buffer A (0.5 M NaCl, 20 mM Tris-HCl pH 7.9) containing 5 mM imidazole. After sonication, the cell debris was removed by centrifugation and the supernatant was incubated with Ni-charged resin. The trapped His-tagged proteins were eluted with buffer A containing 0.5 M imidazole. The sample was dialysed against a buffer containing 20% sucrose, 25 mM Tris-HCl pH 8.0. 1 mM EDTA, 25 mM NaCl, 0.01% NP-40, 1 mM PMSF, 10% glycerol, 1 mM DTT and 2 µg/ml leupeptin.

Electrophoretic mobility shift assay

The electrophoretic mobility shift assay using purified TRF proteins was performed by following the previous report (Zhong et al. 1992). The 32P-labelled oligonucleotide probe ((TTAGGG)28, 1.5 ng was incubated with 100 ng of either TRF1 or TRF2 in a 20 µl reaction mixture containing 20 mM HEPES-KOH (pH 7.9), 150 mM KCl, 1 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 5% glycerol, 4% Ficoll (M.W. 400 000) and 2 µg of HaeIII-cleaved E. coli chromosomal DNA. To test the specificity, the competitor oligonucleotide (non-labelled (TTAGGG)28, 30 ng was added to the reaction mixture. After incubation for 30 min at 22 °C, the reaction mixture was analysed by polyacrylamide gel electrophoresis and subsequent autoradiography.

AFM observation and image analysis

Ten to 20 ng of TRF1 or TRF2 were incubated with ~100 ng of pT2AG3 in a reaction buffer containing 5 mM HEPES (pH 7.4), 2 mM MgCl2, 150 mM KCl for 30 min at 25 °C. After fixation with 0.1% glutaraldehyde for 30 min on ice, the reaction mixture was dropped on to a freshly cleaved mica substrate, which was pretreated with 10 mM spermidine. After 15 min, the mica was washed with water and dried under nitrogen. AFM observation was performed with Nanoscope IIIa (Digital Instruments) in air under Tapping ModeTM. The cantilever (Olympus) was 125 µm in length with ~40 N/m spring constant. The scanning frequency was 1–2 Hz and images were captured with the height mode in a 512 x 512 pixel format. The obtained images were plane-fitted and flattened by the computer program accompanying the imaging module. The Scanning Probe Image Processor program (SPIPTM, Image Metrology) was used to make a birds-eye view image.

Since the dimensions of an AFM image incorporate the tip radius, the real diameter of the protein was calculated using the following formula with double-stranded DNA as a size reference (Nettikadan et al. 1996):

Wp/Wd = (rp/rd)1/2

In which Wp and Wd are the diameters of protein and DNA in AFM images, respectively, and rp and rd are the real diameters of the protein and DNA, respectively. The volume of a protein on the AFM image was calculated as described in previous studies (Schneider et al. 1995, 1998). The estimation of the molecular volume of a protein, based on its molecular weight, was also performed as described in previous studies (Nettikadan et al. 1996; Schneider et al. 1998). Based on this calculation, the molecular volume of the TRF2 monomer was estimated to be 75–85 nm3.


   Acknowledgements
 
This study was supported by the Special Co-ordination Funds and the COE Research Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (for K.T). S.H.Y. was a recipient of the JSPS (Japan Society for the Promotion of Science) predoctoral fellowship.


   Footnotes
 
Communicated by: Fumio Hanaoka

aPresent address: Radiobiology Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-Ku, Tokyo, 104-0045, Japan. Back

* Correspondence: E-mail: yoshimura{at}lif.kyoto-u.ac.jp


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Received: 4 November 2003
Accepted: 22 December 2003




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