Insights into the Dynamics of Specific Telomeric Single-Stranded DNA Recognition by Pot1pN

Abstract

The N-terminal oligonucleotide/oligosaccharide-binding fold domain of the Schizosaccharomyces pombe protection of telomeres 1 (Pot1) protein, Pot1pN (residues 1–187 of full-length Pot1), specifically recognizes telomeric single-stranded DNA (ssDNA) via a complex series of molecular interactions that are punctuated by unusual internucleotide hydrogen bonds. While the structure of ssDNA-bound Pot1pN provides an initial model for understanding how the Pot1pN–ssDNA complex is assembled and how specific nucleotide recognition occurs, further refinement requires knowledge of the ssDNA-free state of Pot1pN and the dynamic changes that accompany the binding of ssDNA. Using NMR strategies, we found that ssDNA-free Pot1pN adopts a similar overall protein backbone topology as ssDNA-bound Pot1pN does. Although the backbone structure remained relatively unchanged, we observed unexpected differential dynamic changes within the ssDNA-binding pockets of Pot1pN upon binding of cognate ssDNA. These studies support a model in which conformational selection and induced fit play important roles in the recognition of ssDNA by Pot1pN. Furthermore, the studies presented here provide a more comprehensive understanding of how specific nucleotide recognition is achieved by the telomere-end protection family of essential proteins.

A Small Molecule Inhibitor of Pot1 Binding to Telomeric DNA

“…1 H- 15 N HSQC experiments are a powerful strategy to assess structural alterations to a protein by monitoring chemical shift changes that report on differences in the chemical environment of the protein backbone. Backbone residue assignments are available for free Pot1pN at pH 6.15 (93, 106). However, as CR precipitates from solution at this pH, we conducted the analysis at higher pH.…”

Targeted Inhibition of Replication Protein A Reveals Cytotoxic Activity, Synergy with Chemotherapeutic DNA-Damaging Agents, and Insight into Cellular Function

“…It is also possible that TDRL-523 does not show a high specificity for RPA and is interacting with other proteins that contain structurally similar OB-fold regions, such telomere end protection proteins (34). Although OB-folds adopt a similar global structure, the lack of sequence similarity between these domains suggests that specific interactions between amino acids and nucleic acids allows for the differential binding to nucleic acids (35). We are currently elucidating the interaction between TDRL-505 and specific amino acids in the A and B DBDs of RPA p70 in order to determine if TDRL-505 can potentially interact with other OB-fold containing proteins.…”

Nonadditivity in the Recognition of Single-Stranded DNA by the Schizosaccharomyces pombe Protection of Telomeres 1 DNA-Binding Domain, Pot1-DBD

“…We find that residues present in Loop 12 , which forms the core of the T3/T4 binding pocket, are strongly perturbed in Pot1-DBD, indicating that the T3/T4 binding pocket is altered in the context of Pot1-DBD (Figure 4A). Dynamics studies of isolated Pot1pN indicate that Loop 12 is unstructured in the absence of bound oligonucleotide and formation of the T3/T4 binding pocket involves the co-folding of Loop 12 and d(GGTTAC) (32). These results support a model in which the T3/T4 binding pocket in Pot1-DBD is present in an alternate conformation that no longer provides for the specificity-dictating interactions between the phosphodiester backbones of T3/T4 with A5 (Supplemental Figure 1A).…”