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，包括无法的 半保守的DNA复制机制完全复制线性分子的末端。这是解决的 在酶端粒酶的几乎所有真核生物中，通过在 染色体结束。广泛认为端粒酶的主要作用部位是单链 DNA复制完成后形成的染色体末端的G富含g。一个新开发 测定说监视自发显示器叉在一个间隙端粒区的倒塌已在此证明 是野生型酵母中端粒酶的第二个底物，这是复制过程中产生的折叠叉 双链端粒DNA的。新近折叠的叉子在单个细胞中被端粒酶广泛拉长 除法，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 倒叉。此外，端粒酶伸长新折叠的叉子的能力取决于叉 重塑蛋白。同时，重新检查了端粒型RPA样复合物的作用（t- RPA）在萌芽的酵母中认为，这种复合物促进了双链DNA期间滞后的链合成 复制，而不是保护端粒免受不受管制的切除。其他数据认为这 复合物与规范RPA复合物合作，以稳定双层端粒过程中的复制叉 DNA复制。总的来说，这些观察结果为当前模型提供了重大挑战 端粒稳态保持在野生型酵母中。该应用程序测试了模型的活动 端粒酶响应自发叉塌陷是端粒长度调节的主要决定因素。 AIM 1将检验以下假设，即新近倒塌的叉子在调节中进行的端粒酶活性进行 途径不同于端粒酶如何参与完全复制的染色体末端。 AIM 2将测试 假设两个RPA复合物，一种用于领先链（RPA）和另一个（T-RPA）结合的假设 到滞后，在复制双层端粒复制过程中合作以促进叉子的稳定 脱氧核糖核酸。第三个目标将检查规范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。