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: 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.
Affiliation: Department of Chemistry and Biochemistry, 596 UCB, University of Colorado Boulder, Boulder, CO 80309, USA.