Bah Amadou, Bachand François, Clair Eveline, Autexier Chantal, Wellinger Raymund J
Department of Microbiology and Infectiology, ARN/RNA Group, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Canada.
Nucleic Acids Res. 2004 Mar 26;32(6):1917-27. doi: 10.1093/nar/gkh511. Print 2004.
The maintenance of telomeric repeat DNA depends on an evolutionarily conserved reverse trans criptase called telomerase. In vitro, only the catalytic subunit and a telomerase-associated RNA are required for the synthesis of species-specific repeat DNA. In an attempt to establish a heterologous system for the study of the human telomerase enzyme, we expressed the two core components and predicted regulatory subunits in the yeast Saccharomyces cerevisiae. We show that adequate substrates for human telomerase can be generated; the expressed enzyme was localized in the nucleus and it had the capacity to synthesize human-specific repeats in vitro. However, there was no evidence for human telomerase activity at yeast telomeres in vivo. Therefore functional replacement of the yeast telomerase by the human enzyme may require additional human-specific components. We also replaced the template region of the yeast telomerase RNA with one that dictates the synthesis of vertebrate repeats and performed a detailed molecular analysis of the composition of the telomeres upon outgrowth of such strains. The results suggest that vertebrate repeats on yeast telomeres are subject to a very high degree of repeat turnover and show that an innermost tract of 50 bp of yeast repeats are resistant to replacement.
端粒重复DNA的维持依赖于一种名为端粒酶的进化保守逆转录酶。在体外，物种特异性重复DNA的合成仅需要催化亚基和一种端粒酶相关RNA。为了建立一个用于研究人类端粒酶的异源系统，我们在酿酒酵母中表达了这两个核心组分以及预测的调节亚基。我们发现可以生成人类端粒酶的合适底物；所表达的酶定位于细胞核，并且在体外具有合成人类特异性重复序列的能力。然而，在体内酵母端粒处没有人类端粒酶活性的证据。因此，用人类酶功能性替代酵母端粒酶可能需要额外的人类特异性组分。我们还用一个决定脊椎动物重复序列合成的区域替换了酵母端粒酶RNA的模板区域，并对这些菌株生长后端粒的组成进行了详细的分子分析。结果表明，酵母端粒上的脊椎动物重复序列经历了非常高程度的重复序列周转，并且表明酵母重复序列最里面50 bp的片段对替换具有抗性。
