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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

Deletion of 22 amino acids from the interdomain linker does not hinder nucleic acid binding. (A) The 22 amino acids deleted (shaded gray) are structurally undefined in the Pot1pN+6mer and Pot1pC+6mer crystal structures and predicted to be unstructured (43). (B) Representative binding curves for wild-type and linker deletion proteins. Linker deletion has very little effect on Pot1-DBD binding to either 15mer (blue) or 12mer (green) ligands relative to wild-type (red). The slight increase in affinity is likely due to entropic effects.
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Figure 2: Deletion of 22 amino acids from the interdomain linker does not hinder nucleic acid binding. (A) The 22 amino acids deleted (shaded gray) are structurally undefined in the Pot1pN+6mer and Pot1pC+6mer crystal structures and predicted to be unstructured (43). (B) Representative binding curves for wild-type and linker deletion proteins. Linker deletion has very little effect on Pot1-DBD binding to either 15mer (blue) or 12mer (green) ligands relative to wild-type (red). The slight increase in affinity is likely due to entropic effects.

Mentions: The linker between subdomains is unassigned in the Pot1-DBD+15mer spectrum due to either exchange broadening or overlap. Thus, we investigated its role in nucleic acid binding by creating a linker deletion construct. Deletion of 22 of the 25 amino acids that connect Pot1pN and Pot1pC had no negative impact upon binding affinity (Figure 2 and Supplementary Table S2)(43). In fact, affinity for both 12mer and 15mer increases slightly, likely due to a reduction in the entropic penalty of binding. These data suggest that the structurally uncharacterized linker does not play a role in nucleic acid binding, further confirming the validity of the individual subdomain structures.


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)

Deletion of 22 amino acids from the interdomain linker does not hinder nucleic acid binding. (A) The 22 amino acids deleted (shaded gray) are structurally undefined in the Pot1pN+6mer and Pot1pC+6mer crystal structures and predicted to be unstructured (43). (B) Representative binding curves for wild-type and linker deletion proteins. Linker deletion has very little effect on Pot1-DBD binding to either 15mer (blue) or 12mer (green) ligands relative to wild-type (red). The slight increase in affinity is likely due to entropic effects.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Deletion of 22 amino acids from the interdomain linker does not hinder nucleic acid binding. (A) The 22 amino acids deleted (shaded gray) are structurally undefined in the Pot1pN+6mer and Pot1pC+6mer crystal structures and predicted to be unstructured (43). (B) Representative binding curves for wild-type and linker deletion proteins. Linker deletion has very little effect on Pot1-DBD binding to either 15mer (blue) or 12mer (green) ligands relative to wild-type (red). The slight increase in affinity is likely due to entropic effects.
Mentions: The linker between subdomains is unassigned in the Pot1-DBD+15mer spectrum due to either exchange broadening or overlap. Thus, we investigated its role in nucleic acid binding by creating a linker deletion construct. Deletion of 22 of the 25 amino acids that connect Pot1pN and Pot1pC had no negative impact upon binding affinity (Figure 2 and Supplementary Table S2)(43). In fact, affinity for both 12mer and 15mer increases slightly, likely due to a reduction in the entropic penalty of binding. These data suggest that the structurally uncharacterized linker does not play a role in nucleic acid binding, further confirming the validity of the individual subdomain structures.

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