How telomeric protein POT1 avoids RNA to achieve specificity for single-stranded DNA

Abstract

The POT1-TPP1 heterodimer, the major telomere-specific single-stranded DNA-binding protein in mammalian cells, protects chromosome ends and contributes to the regulation of telomerase. The recent discovery of telomeric RNA raises the question of how POT1 faithfully binds telomeric ssDNA and avoids illicit RNA binding that could result in its depletion from telomeres. Here we show through binding studies that a single deoxythymidine in a telomeric repeat dictates the DNA versus RNA discrimination by human POT1 and mouse POT1A. We solve the crystal structure of hPOT1 bound to DNA with a ribouridine in lieu of the critical deoxythymidine and show that this substitution results in burying the 2(')-hydroxyl group in a hydrophobic region (Phe62) of POT1 in addition to eliminating favorable hydrogen-bonding interactions at the POT1-nucleic acid interface. At amino acid 62, Phe discriminates against RNA binding and Tyr allows RNA binding. We further show that TPP1 greatly augments POT1's discrimination against RNA.

Fig. 1.

Effect of ribonucleotide substitution on POT1-ssDNA binding. EMSA of mPOT1A (A) and hPOT1 (B) with ³²P-labeled dodecameric ssDNA-RNA mixed oligonucleotides of telomeric sequence. Binding mixtures contained 1 nM ³²P-labeled oligonucleotides and 1.5 nM mPOT1A (A) or 20 nM ³²P-labeled oligonucleotides and 40 nM hPOT1 (B). The sequences of the oligonucleotides are detailed in Table 1. (C) Filter-binding experiments of mPOT1A with ³²P-labeled ssDNA-RNA of the indicated sequence (ribonucleotides are depicted in red) were done in duplicate, and the mean of the fraction of mPOT1A-bound ssDNA-RNA was plotted against mPOT1A concentration. Error bars represent the standard deviation of the two measurements.

Fig. 2.

Structural basis for RNA discrimination by hPOT1. (A) A stereo view showing the electron density (2F_o - F_c) around rU4 contoured at 1σ obtained from rigid body refinement of hPOT1V2-d(TTAGGGTTAG) (PDB: 1XJV) against crystallographic data of hPOT1V2-dTrUd(AGGGTTAG) at 1.8 Å. The final refined model is superimposed on the map as a stick model in Corey–Pauling–Koltun (CPK) atomic coloring. Density for the O2^′ is clearly defined in the electron density. The polar and stacking interactions involving dT3, rU4/dT4, dA5, and dG6 in the hPOT1V2-dTrUd(AGGGTTAG) (B) and the hPOT1V2-d(TTAGGGTTAG) (PDB: 1XJV) (C) structures are shown. The two-headed red arrow in (B) indicates a van der Waals contact between the O2^′ of rU4 and the Phe62 of hPOT1. (D–F) hPOT1V2-dTrUd(AGGGTTAG) (red) and hPOT1V2-d(TTAGGGTTAG) (green) structures were superposed to highlight differences in the nucleic acid backbone along nucleotides dT3-dG6 (D), dG7-dT9 (E), and dT10-dG12 (F). The double-headed black arrows indicate instances of significant DNA-C2^′–protein proximity (< 4.5 Å). Note that the numbering scheme for the nucleic acid in this paper (and associated PDBs) is G(1)G(2)T(3)T(4)A(5)G(6)G(7)G(8)T(9)T(10)A(11)G(12). We have numbered the DNA strand for hPOT1V2-d(TTAGGGTTAG) according to this scheme, although previous references, including PDB: 1XJV, adopt the following numbering scheme: [T(1)T(2)A(3)G(4)G(5)G(6)T(7)T(8)A(9)G(10)].

Fig. 3.

Hydrophobicity of Phe62 is critical for RNA discrimination. The crystal structure of hPOT1V2-dTrUd(AGGGTTAG) is shown as a surface representation of the protein and a stick representation of rU4 (A), dG6 (B), dT9 (C), or dG12 (D). The protein is colored according to hydrophobicity of the amino acids [according to the Kyte–Doolittle scale (36)] such that blue to white to dark orange represents increase in hydrophobicity. For clarity, only the pertinent nucleotide (in CPK coloring convention) of the nucleic acid of hPOT1V2-dTrUd(AGGGTTAG) is shown in each panel. The appropriate O2^′, C2^′, and aromatic stacking residues of hPOT1 are indicated. (E–H) Binding data and curve fits for mPOT1A-F62Y with D12 and 4–6R (E), mPOT1A-Y89F with D12 and 4–6R (F), mPOT1A-Y161F with D12 and 7–9R (G), and mPOT1A-Y223F with D12 and 10–12R (H). Binding curves of D12 with mPOT1A mutants are shown as solid blue lines and those with wild-type mPOT1A as dotted blue lines. Binding curves of indicated ribonucleotide-substituted oligonucleotides with mPOT1A mutants are shown as solid red lines and those with wild-type mPOT1A as dotted red lines. Error bars represent the standard deviation of two independent measurements. The ratio of the K_D with a particular oligonucleotide and K_D with D12 for mPOT1A mutants is indicated in black and that for wild-type is indicated in gray.

Fig. 4.

A single ribonucleotide substitution abrogates TPP1’s ability to stimulate POT1-ssDNA binding. Binding curves for mPOT1A-D12 (A), mPOT1A-4R (B), and hPOT1-4R (C) complexes in the presence (blue) and absence (red) of cognate TPP1-N proteins. Error bars represent the standard deviation of two independent measurements. (D) EMSA showing that hTPP1-N binds hPOT1-D12^′ and hPOT1-4R^′ complexes to give distinct bands corresponding to hPOT1-hTPP1-N-D12^′ and hPOT1-hTPP1-N-4R^′, respectively. Note that D12^′ and 4R^′ contain eight deoxythymidines introduced at the 5^′ end of D12 and 4R sequences, respectively, to resolve the POT1-ssDNA versus POT1-TPP1-N-ssDNA complexes.