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The telomeric protein Pot1 from Schizosaccharomyces pombe binds ssDNA in two modes with differing 3' end availability.

Dickey TH, Wuttke DS - Nucleic Acids Res. (2014)

Bottom Line: These experiments reveal one binding mode characterized by only subtle alternations to the individual OB-fold subdomain structures, resulting in an inaccessible 3' end of the ssDNA.The second binding mode, which has equivalent affinity, interacts differently with the 3' end, rendering it available for interaction with other proteins.These findings suggest a structural switch that contributes to telomere end-protection and length regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, 596 UCB, University of Colorado Boulder, Boulder, CO 80309, USA.

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Related in: MedlinePlus

Pot1-DBD forms primarily a 1:1 complex at low concentrations and can form a larger complex at high concentrations in the presence of 12mer, but not 15mer. Amino acid mutation or base substitution can switch between these two binding modes. (A) RI traces (lines) and molar masses calculated by MALS (markers) of 34 μM Pot1-DBD+15mer and 34 μM Pot1-DBD+12mer complexes in 20 mM Tris–HCl pH 8.0, 50 mM NaCl. (B) Increasing the concentration of NaCl to 400 mM causes Pot1-DBD+12mer to elute earlier than Pot1-DBD+15mer and with a variable but larger average molar mass. Protein and DNA are both at 34 μM. (C) Increasing the concentration of Pot1-DBD to 170 μM and increasing the molar ratio to 2:1, Pot1-DBD:oligonucleotide pushes the 12mer-binding mode toward the 2:1 complex. This 12mer-binding mode can be forced to form a 1:1 complex upon 12mer_T9A substitution. Conversely, Pot1-DBD_Y224A mutation or 15mer_G13C substitution can switch the 15mer-binding mode to the 12mer-binding mode. All complexes were injected at 170 μM protein + 85 μM DNA.
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Figure 3: Pot1-DBD forms primarily a 1:1 complex at low concentrations and can form a larger complex at high concentrations in the presence of 12mer, but not 15mer. Amino acid mutation or base substitution can switch between these two binding modes. (A) RI traces (lines) and molar masses calculated by MALS (markers) of 34 μM Pot1-DBD+15mer and 34 μM Pot1-DBD+12mer complexes in 20 mM Tris–HCl pH 8.0, 50 mM NaCl. (B) Increasing the concentration of NaCl to 400 mM causes Pot1-DBD+12mer to elute earlier than Pot1-DBD+15mer and with a variable but larger average molar mass. Protein and DNA are both at 34 μM. (C) Increasing the concentration of Pot1-DBD to 170 μM and increasing the molar ratio to 2:1, Pot1-DBD:oligonucleotide pushes the 12mer-binding mode toward the 2:1 complex. This 12mer-binding mode can be forced to form a 1:1 complex upon 12mer_T9A substitution. Conversely, Pot1-DBD_Y224A mutation or 15mer_G13C substitution can switch the 15mer-binding mode to the 12mer-binding mode. All complexes were injected at 170 μM protein + 85 μM DNA.

Mentions: At lower concentrations (34 μM), Pot1-DBD+12mer and Pot1-DBD+15mer elute with molar masses of ∼45–50 kDa, similar to the predicted molecular weights of 43.2 and 44.1 kDa for the 1:1 Pot1-DBD+12mer and 1:1 Pot1-DBD+15mer complexes, respectively (Figure 3A). Pot1-DBD+15mer elutes as a uniform peak, while Pot1-DBD+12mer elutes as a broad peak, suggesting the presence of multiple conformations or complexes in the presence of 12mer. Despite this mixture of species, the 1:1 complex appears to be far more prevalent than the 2:1 complex based on the calculated molar mass.


The telomeric protein Pot1 from Schizosaccharomyces pombe binds ssDNA in two modes with differing 3' end availability.

Dickey TH, Wuttke DS - Nucleic Acids Res. (2014)

Pot1-DBD forms primarily a 1:1 complex at low concentrations and can form a larger complex at high concentrations in the presence of 12mer, but not 15mer. Amino acid mutation or base substitution can switch between these two binding modes. (A) RI traces (lines) and molar masses calculated by MALS (markers) of 34 μM Pot1-DBD+15mer and 34 μM Pot1-DBD+12mer complexes in 20 mM Tris–HCl pH 8.0, 50 mM NaCl. (B) Increasing the concentration of NaCl to 400 mM causes Pot1-DBD+12mer to elute earlier than Pot1-DBD+15mer and with a variable but larger average molar mass. Protein and DNA are both at 34 μM. (C) Increasing the concentration of Pot1-DBD to 170 μM and increasing the molar ratio to 2:1, Pot1-DBD:oligonucleotide pushes the 12mer-binding mode toward the 2:1 complex. This 12mer-binding mode can be forced to form a 1:1 complex upon 12mer_T9A substitution. Conversely, Pot1-DBD_Y224A mutation or 15mer_G13C substitution can switch the 15mer-binding mode to the 12mer-binding mode. All complexes were injected at 170 μM protein + 85 μM DNA.
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Figure 3: Pot1-DBD forms primarily a 1:1 complex at low concentrations and can form a larger complex at high concentrations in the presence of 12mer, but not 15mer. Amino acid mutation or base substitution can switch between these two binding modes. (A) RI traces (lines) and molar masses calculated by MALS (markers) of 34 μM Pot1-DBD+15mer and 34 μM Pot1-DBD+12mer complexes in 20 mM Tris–HCl pH 8.0, 50 mM NaCl. (B) Increasing the concentration of NaCl to 400 mM causes Pot1-DBD+12mer to elute earlier than Pot1-DBD+15mer and with a variable but larger average molar mass. Protein and DNA are both at 34 μM. (C) Increasing the concentration of Pot1-DBD to 170 μM and increasing the molar ratio to 2:1, Pot1-DBD:oligonucleotide pushes the 12mer-binding mode toward the 2:1 complex. This 12mer-binding mode can be forced to form a 1:1 complex upon 12mer_T9A substitution. Conversely, Pot1-DBD_Y224A mutation or 15mer_G13C substitution can switch the 15mer-binding mode to the 12mer-binding mode. All complexes were injected at 170 μM protein + 85 μM DNA.
Mentions: At lower concentrations (34 μM), Pot1-DBD+12mer and Pot1-DBD+15mer elute with molar masses of ∼45–50 kDa, similar to the predicted molecular weights of 43.2 and 44.1 kDa for the 1:1 Pot1-DBD+12mer and 1:1 Pot1-DBD+15mer complexes, respectively (Figure 3A). Pot1-DBD+15mer elutes as a uniform peak, while Pot1-DBD+12mer elutes as a broad peak, suggesting the presence of multiple conformations or complexes in the presence of 12mer. Despite this mixture of species, the 1:1 complex appears to be far more prevalent than the 2:1 complex based on the calculated molar mass.

Bottom Line: These experiments reveal one binding mode characterized by only subtle alternations to the individual OB-fold subdomain structures, resulting in an inaccessible 3' end of the ssDNA.The second binding mode, which has equivalent affinity, interacts differently with the 3' end, rendering it available for interaction with other proteins.These findings suggest a structural switch that contributes to telomere end-protection and length regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, 596 UCB, University of Colorado Boulder, Boulder, CO 80309, USA.

Show MeSH
Related in: MedlinePlus