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Journal of Biochemistry Advance Access originally published online on December 11, 2006
Journal of Biochemistry 2007 141(1):57-68; doi:10.1093/jb/mvm011
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© 2006 The Japanese Biochemical Society.

Tetraplex Structure of Fission Yeast Telomeric DNA and Unfolding of the Tetraplex on the Interaction with Telomeric DNA Binding Protein Pot1

Hidetaka Torigoe* and Ayako Furukawa

Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan

*To whom correspondence should be addressed: Tel: 81-3-5228-8259, Fax: 81-3-5261-4631, E-mail: htorigoe{at}ch.kagu.tus.ac.jp

Received September 12, 2006; Accepted November 13, 2006


  Abstract

To understand the regulation mechanism of fission yeast telomeric DNA, we analysed the structural properties of Gn: d(GnTTAC) (n = 2–6) and 4Gn: d(GnTTAC)4 (n = 3 and 4), and their interaction with the single-stranded telomeric DNA binding domain of telomere-binding protein Pot1 (Pot1DBD). G4, G5 and G6 formed a parallel tetraplex in contrast with no tetraplex formation by G2 and G3. Also, 4G4 adopted only an antiparallel tetraplex in spite of a mixture of parallel and antiparallel tetraplexes of 4G3. The variety of tetraplex structures was governed by the number of consecutive guanines in a single copy and the number of repeats. The antiparallel tetraplex of 4G4 became unfolded upon the interaction with Pot1DBD. The interaction with mutant Pot1DBD proteins revealed that the ability to unfold the antiparallel tetraplex was strongly correlated with the specific binding affinity for the single-stranded telomeric DNA. The result suggests that the decrease in the free single strand upon the complex formation with Pot1DBD may shift the equilibrium from the tetraplex to the single strand, which may cause the tetraplex unfolding. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex is required for telomerase-mediated telomere regulation.

Key Words: CD spectroscopy, fluorescence resonance energy transfer, telomeric DNA, telomeric DNA binding protein, tetraplex

Abbreviations: EMSA, electrophoretic mobility shift assay; Fam, 6-carboxyfluorescein; FRET, fluorescence resonance energy transfer; GST, glutathione S-transferase; IPTG, isopropyl-ß–D-thiogalactopyranoside; ITC, isothermal titration calorimetry; Pot1DBD, single-stranded telomeric DNA binding domain of fission yeast telomere-binding protein Pot1; WT, wild-type Pot1DBD


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