Analysis of the Binding of the Schizosaccharomyces pombe Protein Protection of Telomeres 1 Telomeric Single-Stranded DNA through the Examination of Specific Amino Acid Mutations

Protection of Telomeres 1 (Pot1) is a protein that binds with high specificity and affinity to the single-stranded DNA overhang of telomeres in yeast and humans. Pot1 serves crucial functions in telomere regulation and in the protection of free telomere ends from catastrophic DNA damage responses. Ongoing research in the lab involves characterization of the fission yeast Schizosaccharomyces pombe Pot1 protein (SpPot1) as a means for understanding the mechanism of how Pot1 binds to telomeres. SpPot1 binds DNA through its DNA-binding domain (Pot1-DBD), which consists of two OB-fold subdomains, Pot1pN and Pot1pC. The interdomain interface between the two subdomains had been proposed to play an important role in shaping the specific binding of Pot1-DBD to telomeric DNA. Additionally, specific residues within the DNA-binding interface of Pot1pN were shown to play critical roles in Pot1-DBD DNA recognition. This thesis project aims to provide a more complete understanding of Pot1 binding by expanding upon studies of both the interdomain and DNA-binding interfaces. Previous data indicated that the interdomain interface makes a significant contribution to DNA binding under high salt conditions. Since salt concentration is known to affect some protein/DNA interactions, the role of the interface was investigated at low salt using specific alanine mutations. Binding studies indicated that the interface has no significant effect on binding at low salt, suggesting a complexity in the binding scheme of SpPot1, which is at least partially salt-dependent. Examination of DNA-binding activity was extended to the DNA-binding interface of Pot1pC. With no structural information available, systematic analysis of server-generated 3D models of Pot1pC was used to predict residues on the binding surface. Alanine mutants were constructed and circular dichroism was used to perform preliminary studies on the structure and stability of the free and DNA-bound mutant proteins compared to wild-type. The data revealed that, while secondary structures seem unaffected by the mutations or by DNA binding, Pot1pC displays an atypical melting curve, indicative of a conformational change during melting