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Cooperative binding of single-stranded t ... in of Schizosaccharomyces pombe
Cooperative binding of single-stranded telomeric DNA by the Pot1 protein of Schizosaccharomyces pombe.
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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 M, Baumann P, Cech TR
Biochemistry
2002-12-10 00:00
41
49
14560-8
Animals,Base Sequence,Binding Sites,Cyclin B,DNA, Fungal,DNA, Single-Stranded,Electrophoretic Mobility Shift Assay,Kinetics,Macromolecular Substances,Oligonucleotides,Oxytricha,Peptide Fragments,Protein Binding,Schizosaccharomyces pombe Proteins,Sodium Chloride,Telomere,Telomere-Binding Proteins,Thermodynamics,Cyclin B,DNA, Fungal,DNA, Single-Stranded,Macromolecular Substances,Oligonucleotides,Peptide Fragments,Schizosaccharomyces pombe Proteins,Telomere-Binding Proteins,pot1 protein, S pombe,Sodium Chloride
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA
Biochemistry
NIGMS GM28039
0006-2960
bi026674z
167
True
12463756
