Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-Cho, Sakyo-Ku, Kyoto 606-8501, Japan.
Genes Cells. 2012 Mar;17(3):186-204. doi: 10.1111/j.1365-2443.2012.01581.x.
In most cancer cells, telomerase is activated to elongate telomere DNA, thereby ensuring numerous rounds of cell divisions. It is thus important to understand how telomerase and the replication fork react with telomeres in human cells. However, the highly polymorphic and repetitive nature of the nucleotide sequences in human subtelomeric regions hampers the precise analysis of sequential events taking place at telomeres in S phase. Here, we have established HeLa cells harboring a single-seeded telomere abutted by a unique subtelomere DNA sequence, which has enabled us to specifically focus on the seeded telomere. We have also developed a modified chromatin immunoprecipitation (ChIP) method that uses restriction digestion instead of sonication to fragment chromatin DNA (RES-ChIP), and a method for immunoprecipitating 5-bromo-2'-deoxyuridine (BrdU)-labeled single-stranded DNA by incubating DNA with anti-BrdU antibody in the nondenaturing condition. We have shown that DNA replication of the seeded telomere takes place during a relatively narrow time window in S phase, and telomerase synthesizes telomere DNA after the replication. Moreover, we have demonstrated that the telomerase catalytic subunit TERT associates with telomeres before telomere DNA replication. These results provide a temporal and spatial framework for understanding DNA replication and telomerase reaction at human telomeres.
在大多数癌细胞中，端粒酶被激活以延长端粒 DNA，从而确保细胞进行多次分裂。因此，了解端粒酶和复制叉如何与人类细胞中的端粒相互作用非常重要。然而，人类端粒区核苷酸序列的高度多态性和重复性阻碍了对 S 期发生的端粒序列事件的精确分析。在这里，我们建立了含有单个端粒的 HeLa 细胞，该端粒由独特的端粒 DNA 序列对接，这使我们能够专门关注种子端粒。我们还开发了一种改良的染色质免疫沉淀 (ChIP) 方法，该方法使用限制酶消化代替超声处理来片段化染色质 DNA（RES-ChIP），以及一种在非变性条件下通过将 DNA 与抗 BrdU 抗体孵育来免疫沉淀 5-溴-2'-脱氧尿苷 (BrdU)-标记的单链 DNA 的方法。我们已经表明，种子端粒的 DNA 复制发生在 S 期的相对较窄的时间窗口内，并且端粒酶在复制后合成端粒 DNA。此外，我们已经证明端粒酶催化亚基 TERT 在端粒 DNA 复制之前与端粒结合。这些结果为理解人类端粒中的 DNA 复制和端粒酶反应提供了时间和空间框架。
