Molecular analyses of telomere regulation mechanism by telomere binding protein Pot1

我们先前已经证明POTL的N-末端区域是一个单链端粒DNA结合域(Pot1DBD)，氨基酸范围从1到182个氨基酸。我们已经建立了大肠杆菌高效表达系统和Pot1DBD的纯化方案。在本研究中，我们分析了纯化的Pot1DBD的三维结构，发现Pot1DBD由3个α-螺旋和8个β链组成，属于OB(寡核苷酸/寡糖结合)折叠家族。6个碱基的单链DNA d(GGTTAC)是与Pot1DBD特异结合的最小端粒DNA序列。我们还用凝胶移位实验分析了一系列突变的Pot1DBD蛋白与端粒DNA d(GGTTAC)的相互作用。S58A、K90A和D125A的结合能力没有明显变化，但…与野生型Pot1DBD相比，D64A、Q120L和K124A的多结合能力显著降低。根据络合物的三维结构判断与d(GGTTAC)接触的氨基酸对形成络合物的贡献大小不同。天然凝胶电泳和CD光谱表明，在150 mM的氯化钠存在下，裂解酵母端粒DNA 4G4：D(G4TTAC)4形成了反平行的四链DNA结构。在4G4的反平行四链中加入Pot1DBD后的CD光谱变化表明，Pot1DBD能够减少反平行四链DNA的数量。此外，在4G4的反平行四链体中加入Pot1DBD后的荧光光谱变化也表明，Pot1DBD能够解开反平行的四链DNA。与一系列突变的Pot1DBD蛋白的相互作用表明，解开反平行四链复合体的能力与单链端粒DNA的特异性结合能力密切相关。这一结果表明，在与Pot1DBD形成络合物时，游离单链的减少可能使平衡从四链向单链移动，从而可能导致四链的展开。另一方面，我们通过酵母双杂交筛选与POTL(POTL C)C-末端区域相互作用的蛋白质。其中一个被分离的候选者是GALL，它是小核仁核糖核蛋白颗粒(SnoRNPs)的一种成分。通过下拉实验和免疫沉淀实验证实POTL C与Garl之间存在直接的体外相互作用。为了分析Garl对端粒长度的影响而构建的ga/-基因敲除突变体是致命的。在该基因敲除突变体中，由于缺少Garl，POTL不能募集端粒酶，这可能导致严重的端粒缩短和致死。这些结果假设POTL可以通过与Garl的相互作用间接地与端粒酶结合。较少

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 … More 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