DNA binding and telomere length regulation of yeast RAP1 homologues.

The repressor activator protein 1 (RAP1) has many important functions in Saccharomyces cerevisiae. At the chromosome ends, it is a negative regulator of telomere length. Here, we show that Saccharomyces castellii/Saccharomyces dairensis telomeric sequences inserted into a S.cerevisiae telomere are counted as part of the telomere, consistent with the presence of high-affinity Rap1p binding sites within these sequences. We show that S.castellii Rap1p (scasRap1p) can regulate telomere length in a S.cerevisiae strain, albeit less stringently. Cloning of the S.dairensis RAP1 homologue (sdaiRAP1) revealed that it encodes the largest RAP1 protein identified to date. Despite its large size, binding analyses of the recombinant sdaiRap1p revealed that the protein binds with the same spacing and with similar affinity to yeast telomeric sequences, as the scer- and scasRAP1 proteins. According to the Rap1p counting model for telomere length regulation, a low density of Rap1p binding sites in a telomere would result in a longer telomere in S.cerevisiae. We have compared the lengths of two individual S.dairensis telomeres and find that their lengths are not regulated to give the same number of high-affinity binding sites. This may be due to other factors than Rap1p having influence on the telomere length regulation.

Analysis of the RAP1 protein binding to homogeneous telomeric repeats in Saccharomyces castellii.

The repressor activator protein 1 (RAP1) plays a role in telomere structure and function inS. cerevisiae. Here, the RAP1 homologue was identified and cloned from the budding yeast Saccharomyces castellii (scasRAP1). The scasRAP1 gene encodes a protein of 826 amino acids and shares an overall high degree of similarity with the S. cerevisiae RAP1 (scerRAP1). We demonstrate that the scasRAP1 is able to complement scerRAP1 in temperature-sensitive S. cerevisiae strains and is able to function as a regulator to maintain the original telomere lengths. Binding analyses of the E. coli-expressed scasRAP1 protein demonstrate that it needs two consecutive telomeric repeats in order to bind the S. castellii telomeric DNA sequences, and that it binds adjacent sites having a 16 bp centre-to-centre spacing. The binding affinity to telomeric DNA of several other yeasts is similar to that of scerRap1p. However, in contrast to scerRap1p, scasRap1p was found to bind the human telomeric sequence. Moreover, the scasRap1p was found to incorporate a variant repeat in its binding to the otherwise homogeneous telomeric DNA of S. castellii. This ability to bind various sites differing in DNA sequence indicates a high degree of adjustability in the binding of scasRap1p to DNA.