UBC Theses and Dissertations

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UBC Theses and Dissertations

Characterization of the S. cerevisiae Rap1 DNA binding domain and biochemical analysis of its interactions with telomeric and silencer DNA Stone, Geoffrey


groove. Deletion of this region resulted in a protein able to bind telomere and silencer DNA with high affinity, suggesting that this region is not essential for DNA binding or HTH1-HTH2 cooperative interaction. Deletion of a "loop" region between HTH1 and HTH2 was also performed, and results suggested a role for this region in optimal binding of Rap 1 to tandemly repeated telomere sequences. Rapl is a multifunctional protein in S. cerevisiae. It plays a role in chromatin organization, gene silencing and transcriptional activation. Activation alone takes place using up to 100 Rapl DNA binding sites on the genome. All of these functions are mediated by Rapl binding to the DNA duplex, and the Rapl DBD structure must be adaptive to the requirements of the various cellular processes. This thesis suggests that the HTH1, HTH2, "tail" and "loop" regions are specifically designed to optimize Rapl function. Cooperative binding to DNA by HTH1-HTH2 allows a certain degree of degeneracy at high affinity binding sites. "Tail" interactions, while not essential for DNA binding, maintain binding site specificity at a "core" ACC sequence. Finally, the "loop" region allows proper alignment of HTH1 and HTH2, increasing the cooperative association of these two motifs.

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