Cooperative binding of single-stranded telomeric DNA by the Pot1 protein of Schizosaccharomyces pombe

Biochemistry. 2002 Dec 10;41(49):14560-8. doi: 10.1021/bi026674z.

Abstract

The fission yeast Pot1 (protection of telomeres) protein is a single-stranded telomeric DNA-binding protein and is required to protect the ends of chromosomes. Its N-terminal DNA-binding domain, Pot1pN, shows sequence similarity to the first OB fold of the telomere-binding protein alpha subunit of Oxytricha nova. The minimal-length telomeric ssDNA required to bind Pot1pN was determined to consist of six nucleotides, GGTTAC, by gel filtration chromatography and filter-binding assay (K(D) = 83 nM). Pot1pN is a monomer, and each monomer binds one hexanucleotide. Experiments with nucleotide substitutions demonstrated that the central four nucleotides are crucial for binding. The dependence of Pot1pN-ssDNA binding on salt concentration was consistent with a single ionic contact between the protein and the ssDNA phosphate backbone, such that at physiological salt condition 83% of the free energy of binding is nonelectrostatic. Subsequent binding experiments with longer ssDNAs indicated that Pot1pN binds to telomeric ssDNA with 3' end preference and in a highly cooperative manner that mainly results from DNA-induced protein-protein interactions. Together, the binding properties of Pot1pN suggest that the protein anchors itself at the very 3' end of a chromosome and then fills in very efficiently, coating the entire single-stranded overhang of the telomere.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites
  • Cyclin B / chemistry
  • Cyclin B / metabolism
  • DNA, Fungal / chemistry
  • DNA, Fungal / metabolism*
  • DNA, Single-Stranded / chemistry
  • DNA, Single-Stranded / metabolism*
  • Electrophoretic Mobility Shift Assay
  • Kinetics
  • Macromolecular Substances
  • Oligonucleotides / chemistry
  • Oligonucleotides / metabolism
  • Oxytricha / chemistry
  • Oxytricha / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Protein Binding
  • Schizosaccharomyces pombe Proteins / chemistry
  • Schizosaccharomyces pombe Proteins / isolation & purification
  • Schizosaccharomyces pombe Proteins / metabolism*
  • Sodium Chloride / chemistry
  • Telomere / chemistry
  • Telomere / metabolism*
  • Telomere-Binding Proteins / chemistry
  • Telomere-Binding Proteins / isolation & purification
  • Telomere-Binding Proteins / metabolism*
  • Thermodynamics

Substances

  • Cyclin B
  • DNA, Fungal
  • DNA, Single-Stranded
  • Macromolecular Substances
  • Oligonucleotides
  • Peptide Fragments
  • Schizosaccharomyces pombe Proteins
  • Telomere-Binding Proteins
  • pot1 protein, S pombe
  • Sodium Chloride