Telomeres present unique challenges for genomes with linear chromosomes, including the inability of the semi-conservative DNA replication machinery to fully duplicate the ends of linear molecules. This is solved in virtually all eukaryotes by the enzyme telomerase, through the addition of telomeric repeats onto chromosome ends. It is widely assumed that the primary site of action for telomerase is the single-stranded G-rich overhang at the ends of chromosomes, formed after DNA replication is complete. We show here that the preferred substrate for telomerase in wild type yeast is instead a collapsed fork generated during replication of duplex telomeric DNA. Furthermore, newly collapsed forks are extensively elongated by telomerase by as much as ~200 nucleotides in a single cell division, indicating that a major source of newly synthesized telomeric repeats in wild type cells occurs at collapsed forks. Fork collapse and the subsequent response by telomerase are coordinated by the dual activities of a telomere-dedicated RPA-like complex, which facilitates replication of duplex telomeric DNA and also recruits telomerase to the fork, thereby ensuring a high probability of re-elongation if DNA replication fails. We further show that the ability of telomerase to elongate newly collapsed forks is dependent on the Rad51 protein, indicating that telomerase activity in response to fork collapse proceeds through a regulatory pathway distinct from how telomerase engages fully replicated chromosome termini. We propose a new model in which spontaneous replication fork collapse and the subsequent response by telomerase is a major determinant of telomere length homeostasis.

中文翻译：

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自发复制叉塌陷调节野生型细胞端粒长度的稳态。

端粒对具有线性染色体的基因组提出了独特的挑战，包括半保守DNA复制机制无法完全复制线性分子的末端。通过将端粒重复序列添加到染色体末端，几乎所有的真核生物都可以通过端粒酶解决该问题。普遍认为端粒酶的主要作用位点是在DNA复制完成后形成的位于染色体末端的单链富含G的突出端。我们在这里显示，野生型酵母中端粒酶的优选底物是双链端粒DNA复制过程中产生的折叠叉。此外，新折叠的叉子在单个细胞分裂中被端粒酶广泛延长了约200个核苷酸，表明在野生型细胞中新合成的端粒重复序列的主要来源发生在折叠的叉子上。端粒专用的RPA样复合物的双重活性可协调前叉的折叠和随后的端粒酶反应，这有利于双链端粒DNA的复制，也可将端粒酶募集到叉中，从而确保了DNA延长断裂的可能性复制失败。我们进一步表明端粒酶延长新折叠的叉子的能力取决于Rad51蛋白，表明端粒酶活性响应叉子崩溃通过不同于端粒酶如何完全复制染色体末端的调节途径进行。