Coordinated interactions of multiple POT1-TPP1 proteins with telomere DNA

Abstract

Telomeres are macromolecular nucleoprotein complexes that protect the ends of eukaryotic chromosomes from degradation, end-to-end fusion events, and from engaging the DNA damage response. However, the assembly of this essential DNA-protein complex is poorly understood. Telomere DNA consists of the repeated double-stranded sequence 5'-TTAGGG-3' in vertebrates, followed by a single-stranded DNA overhang with the same sequence. Both double- and single-stranded regions are coated with high specificity by telomere end-binding proteins, including POT1 and TPP1, that bind as a heterodimer to single-stranded telomeric DNA. Multiple POT1-TPP1 proteins must fully coat the single-stranded telomere DNA to form a functional telomere. To better understand the mechanism of multiple binding, we mutated or deleted the two guanosine nucleotides residing between adjacent POT1-TPP1 recognition sites in single-stranded telomere DNA that are not required for multiple POT1-TPP1 binding events. Circular dichroism demonstrated that spectra from the native telomere sequence are characteristic of a G-quadruplex secondary structure, whereas the altered telomere sequences were devoid of these signatures. The altered telomere strands, however, facilitated more cooperative loading of multiple POT1-TPP1 proteins compared with the wild-type telomere sequence. Finally, we show that a 48-nucleotide DNA with a telomere sequence is more susceptible to nuclease digestion when coated with POT1-TPP1 proteins than when it is left uncoated. Together, these data suggest that POT1-TPP1 binds telomeric DNA in a coordinated manner to facilitate assembly of the nucleoprotein complexes into a state that is more accessible to enzymatic activity.

Keywords: DNA Binding Protein; DNA Structure; G-quadruplex; Nucleotide; POT1; TPP1; Telomerase; Telomeres.

FIGURE 1.

Circular dichroism spectra of single-stranded DNA with varying sequences. A, diagram of three 48-nt DNA templates investigated. hT48wt is comprised of eight hexameric repeats of a wild-type telomere DNA sequence. The hT48GG→CC construct is comprised of eight hexameric repeats of telomeric DNA in which the last two guanosines of each double repeat are mutated to cytidines. hT48→40ΔGG is comprised of eight hexameric repeats of telomeric DNA in which the last two guanosines of each double repeat are deleted. Each construct contains four consecutive POT1-TPP1 recognition sites, as indicated by the lines above each sequence. B, the CD spectra representing the average of three readings for each DNA sequence depicted in A are shown in buffer containing 75 mm NaCl. The hT48wt spectrum is characteristic of antiparallel G-quadruplexes typical of telomeric DNA in the presence of Na⁺. The spectra of hT48GG→CC and hT48→40ΔGG, however, are indicative of DNA adopting B-form helices, the most common ssDNA conformation. C, CD spectra as obtained in B but in the presence of 75 mm KCl. The hT48wt displays features that are characteristic of hybrid-type G-quadruplexes consisting of both parallel and antiparallel strands, known to be the dominant conformation of longer telomere sequences in the presence of K⁺. The spectra for both hT48GG→CC and hT48→40ΔGG lack these features, as both present as consistent with common B-form helices.

FIGURE 2.

Binding of multiple POT1-TPP1 proteins to wild-type and mutant telomere DNA templates. EMSA experiments of hT48wt (A), hT48GG→CC (B), and hT48→40ΔGG (C) constructs, respectively. EMSAs were performed with DNA concentration remaining constant in each lane and POT1-TPP1 concentration increases from 0 – 2× molar excess/POT1 binding site on the DNA template (in 0.2× molar excess increments from left to right). Numbers in A indicate the number of POT1-TPP1 heterodimers bound to the DNA. D, quantification of the EMSA data for POT1-TPP1 binding to the three oligonucleotides described.

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.

FIGURE 4.

DNase I digestion of 48-nt native and mutant telomeric DNA and POT1-TPP1-DNA nucleoprotein complexes. A ssDNA probe with a native hT48wt (A) or mutant hT48GG→CC (B) telomere sequence was 5′ end-labeled and subjected to degradation by DNase I. DNA samples were either incubated with DNase I alone (-POT1-TPP1) or were first coated with POT1-TPP1 protein (+POT1-TPP1). All samples were subjected to degradation by DNase I digestion in a time course from 0–24 h, as indicated. C, the rate of degradation of each DNA 48-nt substrate with and without POT1-TPP1. The entire gels are provided in supplemental Fig. 5.

FIGURE 5.

Schematic representation of telomere complexes and their relative rates of DNase I digestion. The left panel shows a 48-nt strand of native, G-rich telomeric DNA folded into an illustrative G-quadruplex. This DNA formation is highly resistant to DNase I digestion. Both native and mutant ssDNA can be saturated with POT1-TPP1 proteins to form compact nucleoprotein complexes, as indicated in the center panel. The POT1-TPP1-coated DNA structure is degraded by DNase I more quickly than the G-quadruplex DNA but slower than that of unstructured and unbound DNA. Relatively unstructured ssDNA such as hT48GG→CC (right panel) is rapidly degraded by DNase I.