Investigating the DNA binding properties of the Telomere End-Binding Protein Cdc1

Telomeres are the nucleoprotein complexes that protect the ends of linear chromosomes. Telomeres terminate with a 3' single-stranded G-rich overhang sequence, which is specifically recognized by the Telomere End Protection (TEP) family of proteins. The Saccharomyces cerevisiae TEP protein, Cdc13, binds the single-stranded overhang of yeast telomeres through sequence specific interactions with its DNA binding domain (DBD).;While the essential role of Cdc13 is end protection, Cdc13 has also been shown to both positively and negatively regulate telomerase activity in vivo. To directly test the effect of Cdc13 on telomerase activity, in vitro, we designed a minimal, reconstituted system consisting of the catalytic subunit of yeast telomerase, a miniaturized construct of the yeast telomerase RNA, and purified, recombinant Cdc13 proteins, in collaboration with the Cech Laboratory here at the University of Colorado. We found that, unlike the TEP protein in humans, Pot1, Cdc13 effectively inhibits telomerase even when it is bound up to 17 nucleotides away from the 3' end. Our data support a model in which Cdc13(DBD) and Cdc13 occlude the 3' end from telomerase access, with slight differences in the mechanism of inhibition between the two protein constructs.;Biochemical characterization of the full-length Cdc13 protein unexpectedly demonstrated a weaker affinity for cognate telomeric sequence relative to Cdc13(DBD). We examined several reasons for this attenuation in affinity: length preference, sequence specificity, and other protein domains. We found that, like Cdc13(DBD), the full-length protein minimally binds an 11-mer telomeric oligonucleotide, however, the sequence specificity profile between the two proteins reveals that Cdc13 recognizes the 5' end of the sequence less specifically and the 3' end more specifically than Cdc13(DBD). While significant, the specificity distinction is not sufficient to account for the 100-fold difference in affinity between Cdc13 and Cdc13(DBD). The cdc13-5 mutant, lacking the C-terminus of the protein, displayed similar binding affinities to the full-length protein, suggesting that the N-terminus of the protein may be involved in modulating protein-nucleic acid interactions. Another possible explanation for the affinity difference may be correlated to the pI of the proteins leading to an altered electrostatic predisposition for nucleic acid binding.