Fig. 2. Visualization of TRF2 binding to a model telomere template in vitro. (A–D) The model telomere_500bp DNA with a 54 nt TTAGGG-3′ overhang was incubated with human TRF2 produced in insect cells under conditions described in the text and then directly adsorbed to the EM supports followed by washing, air-drying and rotary shadowcasting with tungsten. Molecules arranged into loops (A), with TRF2 bound internally on the 500 bp TTAGGG tract (B) or at one end of the DNA (C) were observed. In addition, synapsis between the ends of two molecules (D) were present. Following incubation with TRF2, aliquots of the sample were treated with psoralen and UV, followed by deproteinization, surface spreading with cytochrome c and rotary shadowcasting with platinum–palladium. Molecules with small loops at one end as well as two DNAs attached at their ends were present (arrows, E and F). Shown in reverse contrast. Bar is equivalent to the length of a 1.0 kb DNA.

Fig. 4. Model of t-loop formation by TRF2. The results support a model in which the initiating step is the assembly of a TRF2 complex at the ss/ds telomeric junction either by direct binding or by sliding from an internal site. Once formed, loops could be generated by one of several routes, two of which are illustrated here. The junction-bound TRF2 complex may fold backwards to bind an internal site on the telomeric duplex to form a loop (left). Alternatively (right), a second TRF2 complex assembled at an internal site may interact with the end-bound complex to form a loop. A scheme involving a sliding clamp from which a loop grows remains a formal possibility, but is not favored by the observation of two molecules joined in trans. While we have shown a sequential assembly of additional TRF2 during loop formation, the masses of protein at each stage remain to be defined more precisely. In both cases, the loop facilitates the invasion of the overhang into the duplex tract to form a t-loop (bottom).

Fig. 1. Model telomere templates and the variants of the model telomere_500bp termini. The model telomere_500bp termini were altered in the orientation of the overhang (1), the length of the overhang (2), the sequence of the 3′-end of the overhang (3) and the sequence of the ss/ds junction (4) (A). A telomere-containing clone was engineered to place a nine repeat telomere tract at the end of the linearized plasmid. Long telomeric duplexes were created from this clone by both unidirectional replication (model telomere_±2kb) (B) and expansive cloning (model telomere_500bp) (C). Unidirectional replication (B) utilizes a single telomeric repeat containing oligonucleotide to extend the duplex tract off the end of the linearized plasmid. The result is a wide range of tract sizes (average 2 kb). To generate the 3′ overhang, the model telomere_±2kb DNA was digested with a 5′–3′ exonuclease. Expansive cloning (C) utilizes continued cycles of cloning a nine repeat insert to generate long tracts of fixed lengths. The longest stable tract achieved is ∼500 bp. The model telomere_500bp template, when linearized, contains a 4 nt 5′ overhang to which an oligonucleotide can be ligated to create a variety of model DNAs (A).

Fig. 3. Visualization of the ends of the model telomere DNA bound by TRF1 and TRF2. Examples of looped DNA molecules generated on the model telomere_500bp DNA with a 54 nt TTAGGG-3′ overhang as described in Figure 2A–C shown at higher magnifications reveal a large oligomeric mass of TRF2 at the loop junction (A–D). Incubation of the same DNA with TRF1 (see text for details) generated DNA molecules with balls or chains of balls at one end (E and F). Shown in reverse contrast. Bar is equivalent to the length of a 500 bp DNA.