Spontaneous replication fork collapse regulates telomere length homeostasis in wild type yeast

项目摘要/摘要： 端粒对具有线性染色体的基因组提出了独特的挑战，包括无法 半保守的DNA复制机制，能够完全复制线形分子的末端。这个问题解决了 在几乎所有真核生物中，通过端粒酶，通过将端粒重复序列添加到 染色体结束。人们普遍认为端粒酶的主要作用部位是单链的 DNA复制完成后，富含G的物质出现在染色体末端。一种新开发的 监测间质端粒束自发性监测叉状塌陷的分析已经证明 是野生型酵母中端粒酶的第二底物，它是复制过程中产生的一个折叠的叉子 双链端粒DNA。新折叠的叉子在单个细胞中被端粒酶广泛拉长 分裂，这表明野生型细胞中新合成的端粒重复序列的主要来源发生在 折断的叉子。此外，端粒酶延长新折叠的叉子的能力依赖于叉子。 重塑蛋白质。同时，对端粒专用的RPA样复合体(t-RPA)的作用进行了重新研究。 在萌芽酵母中的RPA)认为，这种复合体促进了双链DNA中滞后链的合成 复制，而不是保护端粒免受不受调控的切除。其他数据表明，这一点 Complex与规范的RPA Complex合作，在双链端粒过程中稳定复制分叉 DNA复制。总体而言，这些观察结果对当前的模型提出了实质性的挑战 在野生型酵母中端粒保持稳定。此应用程序测试的活动的模型 端粒酶对自发性叉状塌陷的反应是端粒长度调节的主要决定因素。 目标1将检验这样一种假设，即新倒塌的叉子的端粒酶活性是通过调节 不同于端粒酶参与完全复制的染色体末端的途径。目标2将测试 假设两个RPA复合体，一个专用于前导链(RPA)，另一个(t-RPA)结合 对于滞后的链，在复制双链端粒的过程中，合作促进叉子的稳定 DNA第三个目标将研究规范的RPA复合体在调节端粒酶方面的新作用，通过 从酵母到人类高度保守的表面。

Project Summary/Abstract: 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. A newly developed assay that monitors spontaneous monitor fork collapse at an interstitial telomeric tract has demonstrated there is a second substrate for telomerase in wild type yeast, which is a collapsed fork generated during replication of duplex telomeric DNA. Newly collapsed forks are extensively elongated by telomerase in a single cell division, indicating that a major source of newly synthesized telomeric repeats in wild type cells occurs at collapsed forks. Furthermore, the ability of telomerase to elongate newly collapsed forks is dependent on fork remodeling proteins. In parallel, a re-examination of the role of a telomere-dedicated RPA-like complex (t- RPA) in budding yeast argues that this complex facilitates lagging strand synthesis during duplex DNA replication, rather than protecting telomeres in from unregulated resection. Additional data argues that this complex collaborates with the canonical RPA complex to stabilize replication forks during duplex telomeric DNA replication. Collectively, these observations provide a substantial challenge to current models for how telomere homeostasis is maintained in wild type yeast. This application tests the model that the activity of telomerase in response to spontaneous fork collapse is a major determinant of telomere length regulation. Aim 1 will test the hypothesis that telomerase activity at newly collapsed forks proceeds through a regulatory pathway distinct from how telomerase engages fully replicated chromosome termini. Aim 2 will test the hypothesis that two RPA complexes, one dedicated to the leading strand (RPA) and the other (t-RPA) bound to the lagging strand, collaborate to promote stabilization of the fork during replication of duplex telomeric DNA. The third Aim will examine a new role for the canonical RPA complex in regulating telomerase, through surfaces that are highly conserved from yeast to humans.