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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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The 3′ end of 12mer, but not 15mer, is available for protein interaction when bound by Pot1-DBD. (A) Pot1-DBD begins to form a larger complex visible by EMSA in the presence of 12mer (presumably the 2:1 complex observed by SEC-MALS in Figure 3). Presumed complexes present are shown in cartoon. (B) In the presence of 15mer, however, there is no evidence of a larger, supershifted species at high concentrations of Pot1-DBD. Concentrations of Pot1-DBD are 0.34 fM, 3.4 fM, 34 fM, 68 fM, 340 fM, 680 fM, 3.4 pM, 6.8 pM, 34 pM, 68 pM, 340 pM, 680 pM, 6.8 nM, 68 nM and 680 nM. (C) Titration of Pot1pN into a solution of Pot1-DBD+12mer also forms a larger Pot1-DBD+Pot1pN+12mer complex. (D) Pot1pN, however, fails to form a larger complex with Pot1-DBD+15mer. Concentrations of Pot1pN are 0 pM, 200 pM, 400 pM, 2 nM, 4 nM, 20 nM, 40 nM, 200 nM, 400 nM, 2 μM and 4 μM.
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Figure 4: The 3′ end of 12mer, but not 15mer, is available for protein interaction when bound by Pot1-DBD. (A) Pot1-DBD begins to form a larger complex visible by EMSA in the presence of 12mer (presumably the 2:1 complex observed by SEC-MALS in Figure 3). Presumed complexes present are shown in cartoon. (B) In the presence of 15mer, however, there is no evidence of a larger, supershifted species at high concentrations of Pot1-DBD. Concentrations of Pot1-DBD are 0.34 fM, 3.4 fM, 34 fM, 68 fM, 340 fM, 680 fM, 3.4 pM, 6.8 pM, 34 pM, 68 pM, 340 pM, 680 pM, 6.8 nM, 68 nM and 680 nM. (C) Titration of Pot1pN into a solution of Pot1-DBD+12mer also forms a larger Pot1-DBD+Pot1pN+12mer complex. (D) Pot1pN, however, fails to form a larger complex with Pot1-DBD+15mer. Concentrations of Pot1pN are 0 pM, 200 pM, 400 pM, 2 nM, 4 nM, 20 nM, 40 nM, 200 nM, 400 nM, 2 μM and 4 μM.

Mentions: While SEC-MALS cannot be used to quantitatively measure the concentration of Pot1-DBD at which the 2:1 complex forms, these data are consistent with its formation at low μM concentrations of Pot1-DBD. This value is in agreement with EMSAs, in which a supershift begins to form near 1 μM Pot1-DBD in the presence of 12mer (Figure 4A). This supershift is absent in Pot1-DBD+15mer EMSAs, corroborating the SEC-MALS data (Figure 4B). We note that the approximate concentration at which the 2:1 complex is observed (low μM) is well above the KD (2 pM) for Pot1-DBD+12mer (28). Thus, Pot1-DBD+12mer exists predominantly as a 1:1 complex at the concentrations required for ssDNA binding.


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)

The 3′ end of 12mer, but not 15mer, is available for protein interaction when bound by Pot1-DBD. (A) Pot1-DBD begins to form a larger complex visible by EMSA in the presence of 12mer (presumably the 2:1 complex observed by SEC-MALS in Figure 3). Presumed complexes present are shown in cartoon. (B) In the presence of 15mer, however, there is no evidence of a larger, supershifted species at high concentrations of Pot1-DBD. Concentrations of Pot1-DBD are 0.34 fM, 3.4 fM, 34 fM, 68 fM, 340 fM, 680 fM, 3.4 pM, 6.8 pM, 34 pM, 68 pM, 340 pM, 680 pM, 6.8 nM, 68 nM and 680 nM. (C) Titration of Pot1pN into a solution of Pot1-DBD+12mer also forms a larger Pot1-DBD+Pot1pN+12mer complex. (D) Pot1pN, however, fails to form a larger complex with Pot1-DBD+15mer. Concentrations of Pot1pN are 0 pM, 200 pM, 400 pM, 2 nM, 4 nM, 20 nM, 40 nM, 200 nM, 400 nM, 2 μM and 4 μM.
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Figure 4: The 3′ end of 12mer, but not 15mer, is available for protein interaction when bound by Pot1-DBD. (A) Pot1-DBD begins to form a larger complex visible by EMSA in the presence of 12mer (presumably the 2:1 complex observed by SEC-MALS in Figure 3). Presumed complexes present are shown in cartoon. (B) In the presence of 15mer, however, there is no evidence of a larger, supershifted species at high concentrations of Pot1-DBD. Concentrations of Pot1-DBD are 0.34 fM, 3.4 fM, 34 fM, 68 fM, 340 fM, 680 fM, 3.4 pM, 6.8 pM, 34 pM, 68 pM, 340 pM, 680 pM, 6.8 nM, 68 nM and 680 nM. (C) Titration of Pot1pN into a solution of Pot1-DBD+12mer also forms a larger Pot1-DBD+Pot1pN+12mer complex. (D) Pot1pN, however, fails to form a larger complex with Pot1-DBD+15mer. Concentrations of Pot1pN are 0 pM, 200 pM, 400 pM, 2 nM, 4 nM, 20 nM, 40 nM, 200 nM, 400 nM, 2 μM and 4 μM.
Mentions: While SEC-MALS cannot be used to quantitatively measure the concentration of Pot1-DBD at which the 2:1 complex forms, these data are consistent with its formation at low μM concentrations of Pot1-DBD. This value is in agreement with EMSAs, in which a supershift begins to form near 1 μM Pot1-DBD in the presence of 12mer (Figure 4A). This supershift is absent in Pot1-DBD+15mer EMSAs, corroborating the SEC-MALS data (Figure 4B). We note that the approximate concentration at which the 2:1 complex is observed (low μM) is well above the KD (2 pM) for Pot1-DBD+12mer (28). Thus, Pot1-DBD+12mer exists predominantly as a 1:1 complex at the concentrations required for ssDNA binding.

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