Cooperative binding of single-stranded telomeric DNA by the Pot1 protein of Schizosaccharomyces pombe.
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.
Lei
Ming
M
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Baumann
Peter
P
Cech
Thomas R
TR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019926:Cyclin B / Q000737:chemistry / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D007700:Kinetics; D046911:Macromolecular Substances; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D016813:Oxytricha / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000089804:Shelterin Complex; D012965:Sodium Chloride / Q000737:chemistry; D016615:Telomere / Q000737:chemistry / Q000378:metabolism*; D034501:Telomere-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013816:Thermodynamics
D019926:Cyclin B; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D009841:Oligonucleotides; D010446:Peptide Fragments; D029702:Schizosaccharomyces pombe Proteins; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; C499082:pot1 protein, S pombe; D012965:Sodium Chloride