|Budget Amount *help
¥12,800,000 (Direct Cost: ¥12,800,000)
Fiscal Year 2006: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2005: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2004: ¥6,900,000 (Direct Cost: ¥6,900,000)
We previously showed that the N-terminal region of Potl ranging from amino acids 1 to 182 is a single-stranded telomeric DNA-binding domain (Pot1DBD). We already established the E. coli overexpression system and the purification scheme of Pot1DBD. In this study, we have analyzed the three-dimensional structure of the purified Pot1DBD, and found that Pot1DBD was composed of three a-helices and eight β-strands and it belonged to OB (oligonucleotide/ oligosaccharide binding)-fold family.
Next, we analyzed the interaction between a series of mutated single-stranded telomeric DNA with various lengths and base sequences and the wild-type Pot1DBD by gel shift assay. A six base single-stranded DNA, d(GGTTAC), was the minimum telomeric DNA sequence for the specific binding of Pot1DBD. We also analyzed the interaction between a series of mutant Pot1DBD proteins and the telomeric DNA, d(GGTTAC), by gel shift assay. The binding ability of S58A, K90A and D125A was not significantly changed, but the
binding ability of D64A, Q120L and K124A was significantly reduced in comparison with that of the wild-type Pot1DBD. The contribution to the complex formation was quite different in magnitude among the amino acids judged to contact with d(GGTTAC) by the three-dimensional structure of the complex.
Native gel electrophoresis and CD spectroscopy revealed that a fission yeast telomeric DNA, 4G4: d(G4TTAC)4, formed an antiparallel tetraplex DNA structure in the presence of 150 mM NaCl. The CD spectral change upon the addition of Pot1DBD to the antiparallel tetraplex of 4G4 indicated that Pot1DBD was able to reduce the amount of the antiparallel tetraplex DNA. Also, the fluorescence spectral change upon the addition of Pot1DBD to the antiparallel tetraplex of 4G4 attached by fluorophore and quencher to the 5' and 3' termini, respectively, showed that Pot1DBD was able to unfold the antiparallel tetraplex DNA. The interaction with a series of 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.
On the other hand, we explored the proteins to interact with the C-terminal region of Potl (Potl C) by yeast two-hybrid screening using S. pombe cDNA library. One of the isolated candidates was Garl, a component of small nucleolar ribonucleoprotein particles (snoRNPs). The direct in vitro interaction between Potl C and Garl was confirmed by pull-down and immunoprecipitation assays. The gar/-knockout mutant constructed to analyze the effect of Garl on the telomere length was lethal. In the knockout mutant, Potl was not able to recruit telomerase due to the absence of Garl, which may cause severe telomere shortening and lethality. These results hypothesize that Potl can associate with telomerase indirectly through the interaction with Garl. Less