Reconstitution of human telomerase activity in vitro
Reconstitution of human telomerase activity in vitro
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DOI：
10.1016/s0960-9822(98)70067-3
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发表时间：
1998-01-29
期刊：
影响因子：
9.2
通讯作者：
Harrington, L
中科院分区：
文献类型：
--
作者：
Beattie, TL;Zhou, W;Harrington, L
端粒酶是一种核糖核蛋白酶复合物，它将单链端粒DNA添加到染色体末端[1]。端粒酶的RNA组分包含端粒DNA添加的模板，对端粒酶的活性至关重要[1,2]。端粒酶蛋白已在纤毛虫、酵母和哺乳动物中发现[3-12]。在酿酒酵母中，Est2蛋白与Euplotes端粒酶的123 kDa逆转录酶亚基同源，对端粒酶活性[8]至关重要。在人类中，端粒酶活性与端粒酶RNA hTR[13]、端粒酶RNA结合蛋白TP1/TLP1[5,12]和人类EST2同源物[12]编码的TP2蛋白（也称为TRT1、hEST2或TCS1[9-11]）有关。然而，足以进行活动的最小复杂度是未知的。我们在网织红细胞裂解物中重建了人类端粒酶的活性，发现体外端粒酶活性只需要外源性hTR和TP2。TP2对端粒酶RNA的识别具有物种特异性，hTR的核苷酸10-159足以维持端粒酶的活性。从含有hTR和重组TP2的网织细胞裂解液中免疫沉淀的端粒酶活性，在TP2逆转录酶结构域中取代保守氨基酸残基完全消除端粒酶活性。我们认为TP2和hTR可能代表了人类端粒酶的最小催化核心。(C)现代生物有限公司
Telomerase is a ribonucleoprotein enzyme complex that adds single-stranded telomere DNA to chromosome ends [1]. The RNA component of telomerase contains the template for telomeric DNA addition and is essential for activity [1,2]. Telomerase proteins have been identified in ciliates, yeast and mammals [3-12]. In Saccharomyces cerevisiae, the Est2 protein is homologous to the 123 kDa reverse transcriptase subunit of Euplotes telomerase, and is essential for telomerase activity [8]. In humans, telomerase activity is associated with the telomerase RNA hTR [13], the telomerase RNA-binding protein TP1/TLP1 [5,12] and the TP2 protein encoded by the human EST2 homolog [12] (also known as TRT1, hEST2 or TCS1 [9-11]). The minimal complex sufficient for activity is, however, unknown. We have reconstituted human telomerase activity in reticulocyte lysates and find that only exogenous hTR and TP2 are required for telomerase activity in vitro. Recognition of telomerase RNA by TP2 was species specific, and nucleotides 10-159 of hTR were sufficient for telomerase activity. Telomerase activity immunoprecipitated from the reticulocyte lysate contained hTR and recombinant TP2, Substitution of conserved amino acid residues in the reverse transcriptase domain of TP2 completely abolished telomerase activity. We suggest that TP2 and hTR might represent the minimal catalytic core of human telomerase. (C) Current Biology Ltd.