Specific interactions of the telomeric protein Rap1p with nucleosomal binding sites

J Mol Biol. 2001 Mar 9;306(5):903-13. doi: 10.1006/jmbi.2001.4458.

Abstract

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-specific, DNA-binding protein which, besides participating in the regulation of telomeres structure and length, is also involved in transcriptional regulation of genes essential for cell growth and in silencing. Whereas the long tracts 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, nucleosomes have been found also in Tetrahymena thermophila telomeres, suggesting that, in general, telomere structural differences between lower and higher eukaryotes could be quantitative, rather than qualitative. Using an in vitro model system, we have addressed the question of whether Rap1p can form a stable ternary complex with nucleosomes containing telomeric binding sites, or competes with nucleosome core formation. The approach we have taken is to place a single Rap1p-binding site at different positions within a nucleosome core and then test the binding of Rap1p and its DNA-binding domain (Rap1p-DBD). We show here that both proteins are able to specifically recognize their nucleosomal binding site, but that binding is dependent on the location of the site within the nucleosome core structure. These results show that 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.

MeSH terms

  • Binding Sites*
  • DNA Footprinting
  • DNA, Fungal / chemistry
  • DNA, Fungal / genetics
  • DNA, Fungal / metabolism*
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism*
  • Deoxyribonuclease I / metabolism
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / metabolism*
  • Models, Molecular
  • Nucleic Acid Conformation
  • Nucleosomes / metabolism*
  • Potassium Permanganate
  • Protein Conformation
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae Proteins*
  • Telomere / metabolism*
  • Telomere-Binding Proteins*
  • Transcription Factors*
  • Transcriptional Activation

Substances

  • DNA, Fungal
  • DNA-Binding Proteins
  • Fungal Proteins
  • Nucleosomes
  • RAP1 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Telomere-Binding Proteins
  • Transcription Factors
  • Potassium Permanganate
  • Deoxyribonuclease I