Specific interactions of the telomeric protein Rap1p with nucleosomal binding sites

The telomeres of Saccharomyces cerevisiae are structurally and functionally well characterized. Their telomeric DNA is packaged by the protein Rap1p ( repressor activator protein 1). Rap1p is a multifunctional, sequence-specif ic, DNA-binding protein which, besides participating in the regulation of t elomeres structure and length, is also involved in transcriptional regulati on of genes essential for cell growth and in silencing. Whereas the long tr acts of telomeric DNA repeats of higher eukaryotes are mostly organized in closely spaced canonical nucleosomal arrays, it has been proposed that the 300 base-pairs of S. cerevisiae telomeric DNA are organized in a large non- nucleosomal structure that has been called the telosome. Recently, nucleoso mes have been found also in Tetrahymena thermophila telomeres, suggesting t hat, in general, telomere structural differences between lower and higher e ukaryotes could be quantitative, rather than qualitative. Using an in vitro model system, we have addressed the question of whether Rap1p can form a s table ternary complex with nucleosomes containing telomeric binding sites, or competes with nucleosome core formation. The approach we have taken is t o place a single Rap1p-binding site at different positions within a nucleos ome core and then test the binding of Rap1p and its DNA-binding domain (Rap 1p-DBD). We show here that both proteins are able to specifically recognize their nucleosomal binding site, but that binding is dependent on the locat ion of the site within the nucleosome core structure. These results show th at a ternary complex between a nucleosome and Rap1p is stable and could be a possible intermediate between telomeric nucleosomes and telosomes in the dynamics of S. cerevisiae telomere organization. (C) 2001 Academic Press.