Ohki R, Tsurimoto T, Ishikawa F
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Mol Cell Biol. 2001 Sep;21(17):5753-66. doi: 10.1128/MCB.21.17.5753-5766.2001.
The end replication problem hypothesis proposes that the ends of linear DNA cannot be replicated completely during lagging strand DNA synthesis. Although the idea has been widely accepted for explaining telomere attrition during cell proliferation, it has never been directly demonstrated. In order to take a biochemical approach to understand how linear DNA ends are replicated, we have established a novel in vitro linear simian virus 40 DNA replication system. In this system, terminally biotin-labeled linear DNAs are conjugated to avidin-coated beads and subjected to replication reactions. Linear DNA was efficiently replicated under optimized conditions, and replication products that had replicated using the original DNA templates were specifically analyzed by purifying bead-bound replication products. By exploiting this system, we showed that while the leading strand is completely synthesized to the end, lagging strand synthesis is gradually halted in the terminal approximately 500-bp region, leaving 3' overhangs. This result is consistent with observations in telomerase-negative mammalian cells and formally demonstrates the end replication problem. This study provides a basis for studying the details of telomere replication.
末端复制问题假说提出，在滞后链DNA合成过程中，线性DNA的末端无法完全复制。尽管这一观点已被广泛接受用于解释细胞增殖过程中的端粒磨损，但从未得到直接证明。为了采用生化方法来理解线性DNA末端是如何复制的，我们建立了一种新型的体外线性猿猴病毒40 DNA复制系统。在这个系统中，末端生物素标记的线性DNA与抗生物素蛋白包被的珠子结合，并进行复制反应。在优化条件下，线性DNA能够高效复制，并且通过纯化与珠子结合的复制产物，特异性地分析了使用原始DNA模板进行复制的产物。利用这个系统，我们发现，虽然前导链能完全合成至末端，但滞后链合成在末端大约500碱基对区域逐渐停止，留下3'端突出。这一结果与在端粒酶阴性哺乳动物细胞中的观察结果一致，并正式证明了末端复制问题。本研究为研究端粒复制的细节提供了基础。
