Fulton T B, Blackburn E H
Departments of Microbiology and Immunology & Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94143-0414, USA.
Mol Cell Biol. 1998 Sep;18(9):4961-70. doi: 10.1128/MCB.18.9.4961.
Telomeres in the budding yeast Kluyveromyces lactis consist of perfectly repeated 25-bp units, unlike the imprecise repeats at Saccharomyces cerevisiae telomeres and the short (6- to 8-bp) telomeric repeats found in many other eukaryotes. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which uses a portion of its RNA moiety as a template. K. lactis telomerase RNA, encoded by the TER1 gene, is approximately 1.3 kb long and contains a 30-nucleotide templating domain, the largest ever examined. To examine the mechanism of polymerization by this enzyme, we identified and analyzed telomerase activity from K. lactis whole-cell extracts. In this study, we exploited the length of the template and the precision of copying by K. lactis telomerase to examine primer elongation within one round of repeat synthesis. Under all in vitro conditions tested, K. lactis telomerase catalyzed only one round of repeat synthesis and remained bound to reaction products. We demonstrate that K. lactis telomerase polymerizes along the template in a discontinuous manner and stalls at two specific regions in the template. Increasing the amount of primer DNA-template RNA complementarity results in stalling, suggesting that the RNA-DNA hybrid is not unpaired during elongation in vitro and that lengthy duplexes hinder polymerization through particular regions of the template. We suggest that these observations provide an insight into the mechanism of telomerase and its regulation.
与酿酒酵母端粒处不精确的重复序列以及许多其他真核生物中发现的短（6至8个碱基对）端粒重复序列不同，芽殖酵母乳酸克鲁维酵母中的端粒由完全重复的25个碱基对单元组成。端粒DNA由核糖核蛋白端粒酶合成，该酶将其RNA部分的一部分用作模板。由TER1基因编码的乳酸克鲁维酵母端粒酶RNA长约1.3 kb，包含一个30个核苷酸的模板结构域，这是迄今为止所检测到的最大的模板结构域。为了研究这种酶的聚合机制，我们从乳酸克鲁维酵母全细胞提取物中鉴定并分析了端粒酶活性。在本研究中，我们利用乳酸克鲁维酵母端粒酶模板的长度和复制的精确性来研究一轮重复序列合成中的引物延伸。在所有测试的体外条件下，乳酸克鲁维酵母端粒酶仅催化一轮重复序列合成，并与反应产物保持结合。我们证明，乳酸克鲁维酵母端粒酶沿模板以不连续的方式聚合，并在模板中的两个特定区域停滞。增加引物DNA-模板RNA互补性的量会导致停滞，这表明在体外延伸过程中RNA-DNA杂交体不会解链，并且长双链体通过模板的特定区域阻碍聚合。我们认为，这些观察结果为端粒酶的机制及其调控提供了深入了解。
