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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

Disruption of the interaction between Pot1pC and the 3′ end of 15mer switches Pot1-DBD to the 12mer-binding mode. (A) In the Pot1pC+9mer complex, nucleotides G7 and T9 of 9mer (corresponding to G13 and T15 in 15mer) form an extended aromatic stack with Trp223 and Tyr224 of Pot1pC/Pot1DBD (PDB ID: 4HIK). Pot1pC is colored green, 9mer is colored violet and atoms are colored by element. (B) Y224A mutation disrupts binding of Pot1pC to 9mer by nearly 10-fold. (C) The same Y224A mutation in Pot1-DBD (blue) has no effect on affinity relative to wild-type Pot1-DBD (red). (D) However, Y224A mutation apparently switches to the 12mer-binding mode based on the supershift (red arrow) at high protein concentration. G13C substitution also creates a supershift indicative of the 12mer-binding mode.
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Figure 5: Disruption of the interaction between Pot1pC and the 3′ end of 15mer switches Pot1-DBD to the 12mer-binding mode. (A) In the Pot1pC+9mer complex, nucleotides G7 and T9 of 9mer (corresponding to G13 and T15 in 15mer) form an extended aromatic stack with Trp223 and Tyr224 of Pot1pC/Pot1DBD (PDB ID: 4HIK). Pot1pC is colored green, 9mer is colored violet and atoms are colored by element. (B) Y224A mutation disrupts binding of Pot1pC to 9mer by nearly 10-fold. (C) The same Y224A mutation in Pot1-DBD (blue) has no effect on affinity relative to wild-type Pot1-DBD (red). (D) However, Y224A mutation apparently switches to the 12mer-binding mode based on the supershift (red arrow) at high protein concentration. G13C substitution also creates a supershift indicative of the 12mer-binding mode.

Mentions: The consistent 1:1 stoichiometry of Pot1-DBD+15mer and the structural similarity of the complex to the individual subdomains suggest a binding mode that fully engages the 3′ end. This occlusion of the 3′ Pot1pN-binding site prevents a second Pot1-DBD molecule from binding, unlike the 12mer-binding mode. Indeed, the crystal structure of Pot1pC+9mer reveals an extended aromatic stack involving two amino acid side chains (Trp223 and Tyr224) and two 3′ bases (G7 and T9) (27) (Figure 5A). Furthermore, based on the conserved chemical shifts in the 15N-HSQC spectra, this structural feature is maintained in the Pot1-DBD+15mer complex (Figure 1).


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)

Disruption of the interaction between Pot1pC and the 3′ end of 15mer switches Pot1-DBD to the 12mer-binding mode. (A) In the Pot1pC+9mer complex, nucleotides G7 and T9 of 9mer (corresponding to G13 and T15 in 15mer) form an extended aromatic stack with Trp223 and Tyr224 of Pot1pC/Pot1DBD (PDB ID: 4HIK). Pot1pC is colored green, 9mer is colored violet and atoms are colored by element. (B) Y224A mutation disrupts binding of Pot1pC to 9mer by nearly 10-fold. (C) The same Y224A mutation in Pot1-DBD (blue) has no effect on affinity relative to wild-type Pot1-DBD (red). (D) However, Y224A mutation apparently switches to the 12mer-binding mode based on the supershift (red arrow) at high protein concentration. G13C substitution also creates a supershift indicative of the 12mer-binding mode.
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Figure 5: Disruption of the interaction between Pot1pC and the 3′ end of 15mer switches Pot1-DBD to the 12mer-binding mode. (A) In the Pot1pC+9mer complex, nucleotides G7 and T9 of 9mer (corresponding to G13 and T15 in 15mer) form an extended aromatic stack with Trp223 and Tyr224 of Pot1pC/Pot1DBD (PDB ID: 4HIK). Pot1pC is colored green, 9mer is colored violet and atoms are colored by element. (B) Y224A mutation disrupts binding of Pot1pC to 9mer by nearly 10-fold. (C) The same Y224A mutation in Pot1-DBD (blue) has no effect on affinity relative to wild-type Pot1-DBD (red). (D) However, Y224A mutation apparently switches to the 12mer-binding mode based on the supershift (red arrow) at high protein concentration. G13C substitution also creates a supershift indicative of the 12mer-binding mode.
Mentions: The consistent 1:1 stoichiometry of Pot1-DBD+15mer and the structural similarity of the complex to the individual subdomains suggest a binding mode that fully engages the 3′ end. This occlusion of the 3′ Pot1pN-binding site prevents a second Pot1-DBD molecule from binding, unlike the 12mer-binding mode. Indeed, the crystal structure of Pot1pC+9mer reveals an extended aromatic stack involving two amino acid side chains (Trp223 and Tyr224) and two 3′ bases (G7 and T9) (27) (Figure 5A). Furthermore, based on the conserved chemical shifts in the 15N-HSQC spectra, this structural feature is maintained in the Pot1-DBD+15mer complex (Figure 1).

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