Identification and characterization of a putative telomere end-binding protein from Tetrahymena thermophila.
Identification and characterization of a putative telomere end-binding protein from Tetrahymena thermophila.
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嗜热四膜虫的假定端粒末端结合蛋白的鉴定和表征。
DOI：
10.1128/mcb.15.3.1144
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发表时间：
1995
影响因子：
5.3
通讯作者：
Henderson,E
中科院分区：
文献类型：
--
作者：
Sheng,H;Hou,Z;Schierer,T;Dobbs,DL;Henderson,E
嗜热四膜虫端粒DNA由一段长的（TTGGGG）n双链重复序列和一个单链（TTGGGG）23′突出端组成。我们已经鉴定并表征了一种蛋白质，该蛋白质特异性结合由双链DNA和3′端端粒重复突出组成的合成端粒底物。这种蛋白质被称为TEP（端粒末端结合蛋白）。在TTGGGG突出端重复的第三个位置中从G到A的变化将底物转化为人端粒类似物，并将结合亲和力降低约三倍。将TTGGGG重复序列中的两个G变为C完全消除了结合。然而，四膜虫重复序列的排列对结合只有很小的影响。结合需要与3′突出端相邻的双链体结构，尽管双链体不需要含有端粒重复序列。TEP不与由许多富含G的序列形成的G-四联体DNA结合。TEP对RNA底物的亲和力大大降低。TEP的拷贝数至少为2 × 104/细胞，在不同的细胞生长和发育条件下存在，尽管其水平不同。UV交联实验表明，TEP的表观分子量约为65 kDa。与其他端粒末端结合蛋白不同，TEP对高盐浓度敏感。
Telomeric DNA ofTetrahymena thermophilaconsists of a long stretch of (TTGGGG)ndouble-stranded repeats with a single-stranded (TTGGGG)23′ overhang at the end of the chromosome. We have identified and characterized a protein that specifically binds to a synthetic telomeric substrate consisting of duplex DNA and the 3′ telomeric repeat overhang. This protein is called TEP (telomere end-binding protein). A change from G to A in the third position of the TTGGGG overhang repeat converts the substrate to a human telomere analog and reduces the binding affinity approximately threefold. Changing two G's to C's in the TTGGGG repeats totally abolishes binding. However, permutation of the Tetrahymena repeat sequence has only a minor effect on binding. A duplex structure adjacent to the 3′ overhang is required for binding, although the duplex need not contain telomeric repeats. TEP does not bind to G-quartet DNA, which is formed by many G-rich sequences. TEP has a greatly reduced affinity for RNA substrates. The copy number of TEP is at least 2 × 104per cell, and it is present under different conditions of cell growth and development, although its level varies. UV cross-linking experiments show that TEP has an apparent molecular mass of approximately ~65 kDa. Unlike other telomere end-binding proteins, TEP is sensitive to high salt concentrations.