21st Century Genetics: Mass Spectrometry of Yeast Telomerase.
21st Century Genetics: Mass Spectrometry of Yeast Telomerase.
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DOI：
10.1101/sqb.2015.80.027656
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Zakian VA
中科院分区：
文献类型：
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作者：
Lin KW;Zakian VA
端粒酶是一种特殊的逆转录酶，在几乎所有的真核生物中维持染色体末端。端粒酶的核心由端粒酶RNA和逆转录酶组成，逆转录酶使用不含RNA的短片段来模板化端粒重复序列的添加。此外，一种或多种辅助蛋白是体内端粒酶作用所必需的。研究得最好的辅助蛋白是Est1，它在酵母和人类中都是保守的。在芽殖酵母中，Est 1有两个关键的体内功能：通过与端粒结合蛋白Cdc13相互作用，它将端粒酶募集到端粒，并且它还增加端粒酶活性。虽然芽殖酵母端粒酶高度受细胞周期调控，但Est1是唯一丰度受细胞周期调控的端粒酶亚基。据报道，近400个酵母基因影响端粒长度，尽管其中大多数的具体功能尚不清楚。为了通过质谱鉴定新的端粒酶调节剂，我们开发了纯化酵母端粒酶及其相关蛋白的方法。我们总结了我们使用的方法和描述的实验表明，四个端粒相关蛋白质鉴定的质谱，其中没有一个已经连接到端粒，影响端粒长度和细胞周期调控端粒酶通过控制EST 1丰度。
Telomerase is a specialized reverse transcriptase that maintains the ends of chromosomes in almost all eukaryotes. The core of telomerase consists of telomerase RNA and the reverse transcriptase that uses a short segment without the RNA to template the addition of telomeric repeats. In addition, one or more accessory proteins are required for telomerase action in vivo. The best-studied accessory protein is Est1, which is conserved from yeasts to humans. In budding yeast, Est1 has two critical in vivo functions: By interaction with Cdc13, a telomere-binding protein, it recruits telomerase to telomeres, and it also increases telomerase activity. Although budding yeast telomerase is highly regulated by the cell cycle, Est1 is the only telomerase subunit whose abundance is cell cycle–regulated. Close to 400 yeast genes are reported to affect telomere length, although the specific function of most of them is unknown. With the goal of identifying novel telomerase regulators by mass spectrometry, we developed methods for purifying yeast telomerase and its associated proteins. We summarize the methods we used and describe the experiments that show that four telomerase-associated proteins identified by mass spectrometry, none of which had been linked previously to telomeres, affect telomere length and cell cycle regulation of telomerase by controlling Est1 abundance.