Mutational analyses of a single-stranded telomeric DNA binding domain of fission yeast pot1: conflict with X-ray crystallographic structure.
To understand the telomere regulation mechanism in relation to cell aging and cancer, we examined the single-stranded telomeric DNA binding domain (ssDBD) of fission yeast telomere-binding protein Pot1 by constructing a series of deletion mutants. We found that Pot1(1-182) (amino acids 1-182) stably expressed in Escherichia coli without any degradation retained a stable folded structure and functional telomeric DNA binding activity, indicating that Pot1(1-182) corresponds to ssDBD. We investigated the amino acids of Pot1(1-182) involved in single-stranded telomeric DNA recognition by constructing a series of site-directed mutants. Although the previously reported X-ray crystallographic structure suggests that 12 amino acids contact the telomeric DNA, an electrophoretic mobility shift assay and isothermal titration calorimetry analyses of the binding ability of the site-directed mutants indicated that only five amino acids significantly contributed to telomeric DNA recognition. We conclude that the contribution to recognition is quite different in magnitude among the amino acids judged to contact the target by X-ray crystallographic structure.
Torigoe
Hidetaka
H
Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Tokyo, Japan. htorigoe@ch.kagu.tus.ac.jp
Dohmae
Naoshi
N
Hanaoka
Fumio
F
Furukawa
Ayako
A
D002151:Calorimetry; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004252:DNA Mutational Analysis; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*; D000089804:Shelterin Complex; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D034501:Telomere-Binding Proteins / Q000235:genetics*; D013816:Thermodynamics
D029702:Schizosaccharomyces pombe Proteins; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; C499082:pot1 protein, S pombe; D004247:DNA