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

自发复制叉崩溃调节野生型酵母的端粒长度稳态

• 批准号: 10371165
• 负责人: Jan Karlseder
• 金额: $ 38万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10371165
• 关键词: Affect; Binding; Biological Assay; Cell Proliferation; Cell division; Cells; Chromosomes; Complex; DNA; DNA biosynthesis; DNA replication fork; Data; Defect; Development; Ensure; Enzymes; Eukaryota; Excision; Frequencies; Genes; Genetic; Genetic Screening; Genome; Genome Stability; Health; Homeostasis; Human; Individual; Lead; Length; Life; Link; Malignant Neoplasms; Mediating; Modeling; Monitor; Mutation; Nucleotides; Phenotype; Play; Process; Proteins; Protocols documentation; Regulation; Regulatory Pathway; Resolution; Role; Saccharomycetales; Site; Source; Specificity; Surface; Telomerase; Telomere Maintenance; Telomere Pathway; Testing; Visit; Yeasts; base; cell type; design; genetic analysis; genome-wide; interstitial; mutant; protein complex; recruit; response; telomere; virtual

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.

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，包括端粒无法 半保守 DNA 复制机制，可完全复制线性分子的末端。这已解决 在几乎所有真核生物中，端粒酶通过将端粒重复序列添加到 染色体末端。人们普遍认为端粒酶的主要作用位点是单链 染色体末端富含 G 的突出端，在 DNA 复制完成后形成。一种新开发的 监测间质端粒束自发监测叉塌陷的测定已证明 是野生型酵母中端粒酶的第二底物，是复制过程中产生的折叠叉 双链体端粒DNA。单细胞中新塌陷的分叉被端粒酶广泛拉长 分裂，表明野生型细胞中新合成的端粒重复的主要来源发生在 折叠的叉子。此外，端粒酶延长新塌陷的分叉的能力取决于分叉 重塑蛋白质。与此同时，重新审视端粒专用的 RPA 样复合物（t- 芽殖酵母中的 RPA）认为该复合物促进双链 DNA 过程中滞后链的合成 复制，而不是保护端粒免受不受管制的切除。额外的数据表明，这 复合物与典型的 RPA 复合物合作，在双链体端粒期间稳定复制叉 DNA复制。总的来说，这些观察结果对当前的模型提出了重大挑战 野生型酵母维持端粒稳态。该应用程序测试了以下活动的模型： 端粒酶响应自发叉塌陷是端粒长度调节的主要决定因素。 目标 1 将检验以下假设：新塌陷的分叉上的端粒酶活性通过监管机制进行。 与端粒酶参与完全复制的染色体末端的方式不同的途径。目标 2 将测试 假设两个 RPA 复合物，一个专用于前导链 (RPA)，另一个 (t-RPA) 结合 与滞后链合作，在双链体端粒复制过程中促进叉的稳定 脱氧核糖核酸。第三个目标将通过以下方式研究经典 RPA 复合体在调节端粒酶中的新作用： 从酵母到人类高度保守的表面。