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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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Comparison of subdomain and Pot1-DBD+15mer spectra shows global similarity except at a putative subdomain interface. (A) The 15N -HSQC spectrum of Pot1-DBD (red) bound to 15mer overlays well with the spectra Pot1pN (blue) and Pot1pC (green) bound to 6mer and 9mer, respectively. (B) An enlarged portion of the superpositioned spectra in (A) shows examples of residues that change chemical environment (dark red arrow) and residues that experience multiple distinct chemical environments in Pot1-DBD+15mer, resulting in peak splitting (orange arrows). (C) Altered residues cluster at a putative subdomain interface when mapped onto crystal structures of Pot1pN+6mer (PDB ID: 1QZH) and Pot1pC+9mer (PDB ID: 4HIK). Amino acids with amides shifted >0.05 ppm are shown as dark red spheres and amino acids with split peaks in Pot1-DBD are shown as orange spheres. Unshifted amino acids are colored blue and green for Pot1pN and Pot1pC, respectively. Amino acids that are unassigned in both Pot1-DBD+15mer and subcomplex spectra are colored gray and amino acids unassigned only in Pot1-DBD are colored yellow. DNA is represented by violet sticks. This panel was created using MacPyMOL (42). See supplementary Table S1 for a list of shifted amino acids.
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Figure 1: Comparison of subdomain and Pot1-DBD+15mer spectra shows global similarity except at a putative subdomain interface. (A) The 15N -HSQC spectrum of Pot1-DBD (red) bound to 15mer overlays well with the spectra Pot1pN (blue) and Pot1pC (green) bound to 6mer and 9mer, respectively. (B) An enlarged portion of the superpositioned spectra in (A) shows examples of residues that change chemical environment (dark red arrow) and residues that experience multiple distinct chemical environments in Pot1-DBD+15mer, resulting in peak splitting (orange arrows). (C) Altered residues cluster at a putative subdomain interface when mapped onto crystal structures of Pot1pN+6mer (PDB ID: 1QZH) and Pot1pC+9mer (PDB ID: 4HIK). Amino acids with amides shifted >0.05 ppm are shown as dark red spheres and amino acids with split peaks in Pot1-DBD are shown as orange spheres. Unshifted amino acids are colored blue and green for Pot1pN and Pot1pC, respectively. Amino acids that are unassigned in both Pot1-DBD+15mer and subcomplex spectra are colored gray and amino acids unassigned only in Pot1-DBD are colored yellow. DNA is represented by violet sticks. This panel was created using MacPyMOL (42). See supplementary Table S1 for a list of shifted amino acids.

Mentions: The crystal structures of the Pot1pN+6mer and Pot1pC+9mer subdomains provide some structural insight into the complete S. pome Pot1-DBD (27,31). However, these structures fail to capture the relative orientation of the two domains, the potential influence the domains may have upon one another and the role of the extended linker between domains. To answer these questions, we created a new Pot1-DBD construct using the optimized C-terminus of Pot1pC (27). This construct, spanning residues 1–339, structurally and biochemically recapitulates the original 1–389 Pot1-DBD (Supplementary Figure S1). Despite the ease of Pot1pC+9mer crystallization, the new Pot1-DBD+15mer complex, while displaying enhanced solubility properties, remained recalcitrant to crystallization. Therefore, in lieu of X-ray crystallography, we assigned and compared 15N-HSQC spectra of Pot1pN+6mer, Pot1pC+9mer and Pot1-DBD+15mer and mapped information on local chemical shift changes onto the available subdomain structures (Figure 1)(42).


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)

Comparison of subdomain and Pot1-DBD+15mer spectra shows global similarity except at a putative subdomain interface. (A) The 15N -HSQC spectrum of Pot1-DBD (red) bound to 15mer overlays well with the spectra Pot1pN (blue) and Pot1pC (green) bound to 6mer and 9mer, respectively. (B) An enlarged portion of the superpositioned spectra in (A) shows examples of residues that change chemical environment (dark red arrow) and residues that experience multiple distinct chemical environments in Pot1-DBD+15mer, resulting in peak splitting (orange arrows). (C) Altered residues cluster at a putative subdomain interface when mapped onto crystal structures of Pot1pN+6mer (PDB ID: 1QZH) and Pot1pC+9mer (PDB ID: 4HIK). Amino acids with amides shifted >0.05 ppm are shown as dark red spheres and amino acids with split peaks in Pot1-DBD are shown as orange spheres. Unshifted amino acids are colored blue and green for Pot1pN and Pot1pC, respectively. Amino acids that are unassigned in both Pot1-DBD+15mer and subcomplex spectra are colored gray and amino acids unassigned only in Pot1-DBD are colored yellow. DNA is represented by violet sticks. This panel was created using MacPyMOL (42). See supplementary Table S1 for a list of shifted amino acids.
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Figure 1: Comparison of subdomain and Pot1-DBD+15mer spectra shows global similarity except at a putative subdomain interface. (A) The 15N -HSQC spectrum of Pot1-DBD (red) bound to 15mer overlays well with the spectra Pot1pN (blue) and Pot1pC (green) bound to 6mer and 9mer, respectively. (B) An enlarged portion of the superpositioned spectra in (A) shows examples of residues that change chemical environment (dark red arrow) and residues that experience multiple distinct chemical environments in Pot1-DBD+15mer, resulting in peak splitting (orange arrows). (C) Altered residues cluster at a putative subdomain interface when mapped onto crystal structures of Pot1pN+6mer (PDB ID: 1QZH) and Pot1pC+9mer (PDB ID: 4HIK). Amino acids with amides shifted >0.05 ppm are shown as dark red spheres and amino acids with split peaks in Pot1-DBD are shown as orange spheres. Unshifted amino acids are colored blue and green for Pot1pN and Pot1pC, respectively. Amino acids that are unassigned in both Pot1-DBD+15mer and subcomplex spectra are colored gray and amino acids unassigned only in Pot1-DBD are colored yellow. DNA is represented by violet sticks. This panel was created using MacPyMOL (42). See supplementary Table S1 for a list of shifted amino acids.
Mentions: The crystal structures of the Pot1pN+6mer and Pot1pC+9mer subdomains provide some structural insight into the complete S. pome Pot1-DBD (27,31). However, these structures fail to capture the relative orientation of the two domains, the potential influence the domains may have upon one another and the role of the extended linker between domains. To answer these questions, we created a new Pot1-DBD construct using the optimized C-terminus of Pot1pC (27). This construct, spanning residues 1–339, structurally and biochemically recapitulates the original 1–389 Pot1-DBD (Supplementary Figure S1). Despite the ease of Pot1pC+9mer crystallization, the new Pot1-DBD+15mer complex, while displaying enhanced solubility properties, remained recalcitrant to crystallization. Therefore, in lieu of X-ray crystallography, we assigned and compared 15N-HSQC spectra of Pot1pN+6mer, Pot1pC+9mer and Pot1-DBD+15mer and mapped information on local chemical shift changes onto the available subdomain structures (Figure 1)(42).

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