FIGURE 3.

Binding of POT1-TPP1 to sequential recognition sites. A, diagram of DNA sequences investigated. Solid bars above the sequence indicate individual POT1 binding sites. Dashed lines indicate a G→C (asterisk) mutation within the POT1 binding site of the DNA. Shown are electrophoretic mobility shift assays of POT1-TPP1 binding to the hT48GG→CC (B), M3 (C), and M4 (D) DNA fragments, respectively. Experiments were performed the same as in Fig. 2, except that protein concentrations were increased to 0–5× molar excess/POT1 binding site in an attempt to fully saturate the DNA with four bound POT1-TPP1 proteins. Densitometry performed on the last five lanes corresponding to 3–5× molar excess POT1-TPP1 per binding site was quantified to reveal the proportion of each bound species for the individual constructs. E, model depicting a simple mechanistic explanation for the cooperative binding observed in the presence of multiple POT1-TPP1 loading events and the process that M3 and M4 mutants obstruct. Step 1, POT1-TPP1 binds preferentially to the most 3′ binding site. Step 2, POT1-TPP1 assumes a conformational change upon binding of an appropriate DNA substrate. Step 3, the conformational change assumed in step 2 facilitates recruitment of an additional POT1-TPP1 heterodimer to the 5′ adjacent binding site. Step 4, the second bound POT1-TPP1 undergoes the DNA-binding dependent conformational change and then facilitates recruitment and binding of additional POT1-TPP1 heterodimers in a sequential 3′→5′ manner until the single-stranded telomeric DNA is fully coated.