RAP1 at the Yeast Telomere

University dissertation from Johan Wahlin, Dep. of Cell and Organism Biology, Sölvegatan 29, 223 62 Lund, Sweden

Abstract: Telomeres are specialized complexes of DNA and proteins that cap and confer stability to the ends of eukaryotic chromosomes. The telomeric DNA of most eukaryotes is composed of tandemly arranged short repeats. Within the budding yeasts these repeats are diverged in length as well as in nucleotide composition. However, they contain a sequence resembling the consensus binding sequence of the major telomere-binding protein Repressor Activator Protein 1 (Rap1p). It is shown here that the telomeric repeat units of several budding yeasts are indeed bound by the Saccharomyces cerevisiae RAP1 protein(scerRap1p) in vitro. Furthermore, the protein can bind to some of those sequences with spatial flexibility. The identification of two novel RAP1 homologues are also presented here. They were isolated from Saccharomyces castellii (scasRAP1) and Saccharomyces dairensis (sdaiRAP1). Sequence analyses revealed that the DNA-binding and C-terminal domains of these proteins are similar to the corresponding regions of scerRap1p, reflecting the importance of these regions in the function of the protein. The similarity between the proteins is underscored by the finding that the scasRAP1 gene can replace the scerRAP1 gene in a S. cerevisiae strain. Binding analyses revealed that the scas- and sdaiRAP1 proteins bind to telomeric sequences in a similar manner as scerRap1p. However unlike scerRap1p, these proteins can bind to the telomeric repeats of vertebrates in vitro. It is shown here that telomeric repeats from S. castellii/S. dairensis introduced into a S. cerevisiae telomere are sensed as being part of the telomere by the telomere length sensing mechanism. Such hybrid telomeres are kept at slightly shorter lengths than telomeres containing S. cerevisiae telomeric DNA only. Since these S. castellii/S. dairensis telomeric repeats have a denser distribution of Rap1p binding sites than S. cerevisiae telomeric DNA, this shortening of the telomeric tracts would be predicted from the RAP1 protein counting model of telomere length regulation.

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