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.

项目概要/摘要： 端粒对具有线性染色体的基因组提出了独特的挑战，包括染色体不能在端粒中表达。 半保守的DNA复制机器完全复制线性分子的末端。这就解决了 在几乎所有的真核生物中，通过端粒酶，通过在端粒重复序列上添加端粒重复序列， 染色体末端人们普遍认为端粒酶的主要作用位点是单链端粒酶。 染色体末端富含G的突出端，在DNA复制完成后形成。新开发的 一项监测自发性监测叉在间质端粒束处塌陷的测定表明， 是野生型酵母中端粒酶的第二底物，其是在复制期间产生的折叠叉 双链端粒DNA在单个细胞中，新折叠的分叉被端粒酶广泛延长 分裂，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 折叠的叉子此外，端粒酶延长新折叠的叉的能力依赖于叉。 重塑蛋白同时，重新检查端粒专用RPA样复合物（t- RPA）在芽殖酵母中的研究认为，这种复合物促进了双链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。更多数据显示， 复合物与典型的RPA复合物合作，以稳定双链端粒过程中的复制叉 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， 端粒稳态在野生型酵母中得以维持。该应用程序测试的模型， 端粒酶对自发性分叉塌陷响应是端粒长度调节的主要决定因素。 目的1将检验新塌陷分叉处的端粒酶活性通过调节性的 与端粒酶如何参与完全复制的染色体末端不同的途径。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 的滞后链，合作，以促进稳定的叉在复制双链端粒 DNA.第三个目标将研究经典RPA复合物在调节端粒酶中的新作用， 从酵母到人类都高度保守的表面。