Jacob N K, Skopp R, Price C M
Department of Molecular Genetics, Biochemistry and Microbiology, School of Medicine, University of Cincinnati, ML0524, 231 Albert Sabin Way, Cincinnati, OH 45267, USA.
EMBO J. 2001 Aug 1;20(15):4299-308. doi: 10.1093/emboj/20.15.4299.
To learn more about the structure of the DNA terminus at Tetrahymena thermophila telomeres, we have devised a ligation-mediated primer extension protocol to accurately measure the length of the G-strand overhang. We show that overhang length and the identity of the 3'-terminal nucleotide are tightly regulated. The majority of overhangs terminate in the sequence 5'-TTGGGGT and >80% are either 14-15 or 20-21 nucleotides in length. No significant changes in overhang length were detected as cells traversed the cell cycle. However, changes in length distribution were observed when cells exited the cell cycle, indicating an altered balance between DNA synthesis and degradation or end protection. We also provide evidence that rDNA molecules have overhangs on both telomeres. Full-length rDNA could be cloned by a strategy that depends on overhangs being present at both ends. Moreover, analysis of leading strand telomeres revealed that a significant fraction have overhangs > or =5 nucleotides. Our results indicate that generation of the terminal telomeric DNA structure is highly regulated and requires several distinct DNA-processing events.
为了更深入了解嗜热四膜虫端粒处DNA末端的结构，我们设计了一种连接介导的引物延伸方案，以精确测量G链悬突的长度。我们发现悬突长度和3'末端核苷酸的身份受到严格调控。大多数悬突以5'-TTGGGGT序列终止，超过80%的悬突长度为14 - 15个或20 - 21个核苷酸。当细胞经历细胞周期时，未检测到悬突长度有显著变化。然而，当细胞退出细胞周期时，观察到长度分布发生了变化，这表明DNA合成与降解或末端保护之间的平衡发生了改变。我们还提供证据表明rDNA分子的两个端粒都有悬突。全长rDNA可以通过一种依赖于两端都存在悬突的策略进行克隆。此外，对前导链端粒的分析表明，相当一部分端粒的悬突长度大于或等于5个核苷酸。我们的结果表明，末端端粒DNA结构的生成受到高度调控，并且需要几个不同的DNA加工事件。
