Cech T R, Lingner J
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Ciba Found Symp. 1997;211:20-8; discussion 28-34. doi: 10.1002/9780470515433.ch3.
Telomerase, the enzyme that extends chromosomal DNA ends in most eukaryotes, contains essential RNA and protein subunits. We have been studying telomere replication in hypotrichous ciliates such as Euplotes aediculatus, which have numerous short macronuclear DNA molecules and therefore are highly enriched in telomeres and in telomerase. Cloning and sequencing genes for the RNA subunits from several ciliates revealed that telomerase RNAs with insignificant nucleotide sequence homology nevertheless form a common secondary structure. Affinity chromatography based on the sequence of the RNA subunit was used to purify the Euplotes telomerase as an active ribonucleoprotein enzyme. Two protein subunits, 123 kDa and 43 kDa, were identified. The finding of a yeast homologue to the 123 kDa subunit suggests that telomerase protein components may be much more highly conserved in evolution than the RNA subunits. The purified Euplotes telomerase has no activity with blunt-ended DNA primers, but instead requires a four to six nucleotide single-stranded 3' tail. This result supports a model for telomere replication in which other activities such as helicases or nucleases activate replicated DNA for extension by telomerase, a model that may be applicable to telomere replication in diverse eukaryotes.
端粒酶是一种在大多数真核生物中延长染色体DNA末端的酶，它包含必需的RNA和蛋白质亚基。我们一直在研究诸如嗜热四膜虫等寡毛纲纤毛虫中的端粒复制，这些纤毛虫有许多短的大核DNA分子，因此端粒和端粒酶高度富集。对几种纤毛虫的RNA亚基基因进行克隆和测序后发现，核苷酸序列同源性不显著的端粒酶RNA却形成了共同的二级结构。基于RNA亚基序列的亲和层析被用于纯化嗜热四膜虫端粒酶，使其成为一种有活性的核糖核蛋白酶。鉴定出了两个蛋白质亚基，分别为123 kDa和43 kDa。发现123 kDa亚基存在酵母同源物，这表明端粒酶蛋白质组分在进化上可能比RNA亚基保守得多。纯化后的嗜热四膜虫端粒酶对平端DNA引物无活性，而是需要一个4至6个核苷酸的单链3'尾。这一结果支持了一种端粒复制模型，即解旋酶或核酸酶等其他活性物质激活复制后的DNA以便由端粒酶进行延伸，该模型可能适用于多种真核生物的端粒复制。
