The telomeric protein Pot1 from Schizosaccharomyces pombe binds ssDNA in two modes with differing 3' end availability.
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.
Affiliation: Department of Chemistry and Biochemistry, 596 UCB, University of Colorado Boulder, Boulder, CO 80309, USA.Show MeSH
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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).
Affiliation: Department of Chemistry and Biochemistry, 596 UCB, University of Colorado Boulder, Boulder, CO 80309, USA.