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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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Model of how Pot1 binding modulates 3′ end accessibility. (A) The electrostatic surface representation of Pot1pC shows an electropositive patch (within the black circle) that may be involved in binding the 3′ end of 12mer (PDB ID: 4HIK). Electrostatic potentials were created using the APBS plug-in in PyMOL with electrostatic potential bounds of 2 ±kT/e (49–51). (B) Pot1-DBD can adopt multiple binding modes that may be important for function. Pot1-DBD binds 15mer as a homogeneous 1:1 complex with an occluded 3′ end. Upon disruption of this binding mode, the complex switches to the 12mer-binding mode, which allows the formation of higher order complexes in vitro and may allow telomerase access in vivo.
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Figure 6: Model of how Pot1 binding modulates 3′ end accessibility. (A) The electrostatic surface representation of Pot1pC shows an electropositive patch (within the black circle) that may be involved in binding the 3′ end of 12mer (PDB ID: 4HIK). Electrostatic potentials were created using the APBS plug-in in PyMOL with electrostatic potential bounds of 2 ±kT/e (49–51). (B) Pot1-DBD can adopt multiple binding modes that may be important for function. Pot1-DBD binds 15mer as a homogeneous 1:1 complex with an occluded 3′ end. Upon disruption of this binding mode, the complex switches to the 12mer-binding mode, which allows the formation of higher order complexes in vitro and may allow telomerase access in vivo.

Mentions: The Pot1-DBD+12mer complex is more challenging to structurally characterize due to its propensity to form a higher molecular weight complex at concentrations necessary for NMR. We have found, however, that Pot1-DBD forms a high-affinity 1:1 complex with 12mer and only forms the 2:1 complex when the concentration of Pot1-DBD is sufficient to compete with the intramolecular interaction between the monomeric Pot1-DBD and the 3′ end of 12mer. This conclusion is supported by the ability of Pot1-DBD to bind 12mer-T9A with low picomolar affinity despite the inability of this oligonucleotide to promote the formation of the 2:1 complex (28). Despite difficulties in characterizing the 1:1 Pot1-DBD+12mer complex, the ability of Pot1-DBD to dimerize on 12mer suggests that the interaction with the 3′ end of 12mer differs significantly from that of 15mer. In contrast, the 5′ end of 12mer and 15mer are identical in sequence and it is likely that their interactions with Pot1pN are more similar. The salt dependence of dimerization suggests that the interaction between Pot1pC and the 3′ end of 12mer has a large electrostatic component, which is consistent with the large number of positively charged resides near the interdomain interface that may participate in binding 12mer (Figure 6A)(49–51). Together, these observations support a model in which the 3′ end of 12mer is tucked into a positively charged pocket of Pot1pC, providing the favorable energetics required for low pM binding. This interaction, however, likely allows fraying of the 3′ end or partial dissociation of Pot1pC, allowing access to the 3′ end.


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)

Model of how Pot1 binding modulates 3′ end accessibility. (A) The electrostatic surface representation of Pot1pC shows an electropositive patch (within the black circle) that may be involved in binding the 3′ end of 12mer (PDB ID: 4HIK). Electrostatic potentials were created using the APBS plug-in in PyMOL with electrostatic potential bounds of 2 ±kT/e (49–51). (B) Pot1-DBD can adopt multiple binding modes that may be important for function. Pot1-DBD binds 15mer as a homogeneous 1:1 complex with an occluded 3′ end. Upon disruption of this binding mode, the complex switches to the 12mer-binding mode, which allows the formation of higher order complexes in vitro and may allow telomerase access in vivo.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4150783&req=5

Figure 6: Model of how Pot1 binding modulates 3′ end accessibility. (A) The electrostatic surface representation of Pot1pC shows an electropositive patch (within the black circle) that may be involved in binding the 3′ end of 12mer (PDB ID: 4HIK). Electrostatic potentials were created using the APBS plug-in in PyMOL with electrostatic potential bounds of 2 ±kT/e (49–51). (B) Pot1-DBD can adopt multiple binding modes that may be important for function. Pot1-DBD binds 15mer as a homogeneous 1:1 complex with an occluded 3′ end. Upon disruption of this binding mode, the complex switches to the 12mer-binding mode, which allows the formation of higher order complexes in vitro and may allow telomerase access in vivo.
Mentions: The Pot1-DBD+12mer complex is more challenging to structurally characterize due to its propensity to form a higher molecular weight complex at concentrations necessary for NMR. We have found, however, that Pot1-DBD forms a high-affinity 1:1 complex with 12mer and only forms the 2:1 complex when the concentration of Pot1-DBD is sufficient to compete with the intramolecular interaction between the monomeric Pot1-DBD and the 3′ end of 12mer. This conclusion is supported by the ability of Pot1-DBD to bind 12mer-T9A with low picomolar affinity despite the inability of this oligonucleotide to promote the formation of the 2:1 complex (28). Despite difficulties in characterizing the 1:1 Pot1-DBD+12mer complex, the ability of Pot1-DBD to dimerize on 12mer suggests that the interaction with the 3′ end of 12mer differs significantly from that of 15mer. In contrast, the 5′ end of 12mer and 15mer are identical in sequence and it is likely that their interactions with Pot1pN are more similar. The salt dependence of dimerization suggests that the interaction between Pot1pC and the 3′ end of 12mer has a large electrostatic component, which is consistent with the large number of positively charged resides near the interdomain interface that may participate in binding 12mer (Figure 6A)(49–51). Together, these observations support a model in which the 3′ end of 12mer is tucked into a positively charged pocket of Pot1pC, providing the favorable energetics required for low pM binding. This interaction, however, likely allows fraying of the 3′ end or partial dissociation of Pot1pC, allowing access to the 3′ end.

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