权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular analyses of telomere regulation mechanism by telomere binding protein Pot1

端粒结合蛋白Pot1对端粒调控机制的分子分析

基本信息

批准号：
16390083
负责人：
TORIGOE Hidetaka
金额：
$ 8.19万
依托单位：
Tokyo University of Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

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

我们以前表明，Potl的N-末端区域从氨基酸1至182是一个单链端粒DNA结合域（Pot 1DBD）。我们已经建立了E。coli表达系统和Pot 1DBD的纯化方案。本研究首先分析了纯化的Pot 1DBD的三维结构，发现Pot 1DBD由3个α-螺旋和8个β-链组成，属于OB（oligonucleotide/ oligosaccharide binding）-fold家族，然后利用凝胶位移分析法分析了一系列不同长度和碱基序列的突变单链端粒DNA与野生型Pot 1DBD的相互作用。6个碱基的单链DNA，d（GGTTAC），是Pot 1DBD特异性结合的最小端粒DNA序列。我们还分析了一系列的突变Pot 1DBD蛋白和端粒DNA，d（GGTTAC）之间的相互作用，通过凝胶迁移试验。S58 A、K90 A和D125 A的结合能力没有显著变化，但其结合能力随时间的延长而降低。 ...更多信息与野生型Pot 1DBD相比，D 64 A、Q120 L和K124 A的结合能力显著降低。复合物形成的贡献是相当不同的幅度之间的氨基酸判断接触d（GGTTAC）的三维结构的complex.Native凝胶电泳和CD光谱显示，一个分裂酵母端粒DNA，4G 4：d（G4 TTAC）4，形成一个反平行的四链体DNA结构在150 mM NaCl的存在下。将Pot 1DBD加入到4G 4的反平行四链体中后的CD光谱变化表明Pot 1DBD能够减少反平行四链体DNA的量。此外，在将Pot 1DBD添加到通过荧光团和猝灭剂分别连接到5'和3'末端的4G 4的反平行四链体时的荧光光谱变化表明Pot 1DBD能够解折叠反平行四链体DNA。与一系列突变Pot 1DBD蛋白的相互作用表明，展开反平行四链体的能力与单链端粒DNA的特异性结合亲和力密切相关。结果表明，与Pot 1DBD形成复合物后，游离单链的减少可能使四链体平衡向单链转移，从而导致四链体解折叠。粟酒酵母cDNA文库其中一个分离的候选人是Garl，小核仁核糖核蛋白颗粒（snoRNP）的组成部分。PotlC和Garl之间的直接体外相互作用通过下拉和免疫沉淀测定来证实。构建的用于分析Garl对端粒长度的影响的gar/-敲除突变体是致死的。在敲除突变体中，由于Garl的缺失，Potl不能募集端粒酶，这可能导致严重的端粒缩短和致死性。这些结果推测Potl可以通过与Garl的相互作用间接与端粒酶结合。少