Regulation of transcription termination by checkpoint kinases Mec1p and Rad53p

检查点激酶 Mec1p 和 Rad53p 对转录终止的调节

• 批准号: 10729762
• 负责人: Ales Vancura
• 金额: $ 49.2万
• 依托单位: ST. JOHN'S UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10729762
• 关键词: ATR gene; Affect; CHEK2 gene; Cell Survival; Cells; Complex; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA damage checkpoint; DNA replication fork; Data; Defect; Deoxyribonucleotides; Environment; Eukaryotic Cell; Excision; Fermentation; Genes; Genetic; Genetic Transcription; Genome Stability; Genomic Instability; Goals; Histones; Lead; Link; Maintenance; Malignant Neoplasms; Meclp; Mediating; Metabolic; Molecular; Mutation; Organism; Pathway interactions; Pattern; Phosphorylation; Phosphotransferases; Poly A; Polyadenylation; Polyadenylation Pathway; RNA Polymerase II; RNA polymerase II largest subunit; Research; Respiration; S phase; Saccharomyces cerevisiae; Signal Transduction; Site; Small Nucleolar RNA; Stress; Testing; Transcriptional Regulation; Untranslated RNA; Yeasts; attenuation; biochemical tools; cancer therapy; cleavage factor; coping; experimental study; gene repression; gene terminator; genotoxicity; innovation; mRNA Cleavage and Polyadenylation Factors; mRNA Precursor; model organism; novel; recruit; replication stress; response; stem; transcription termination; transcriptional reprogramming; tripolyphosphate; undergraduate student

PROJECT SUMMARY The goal of this project is to understand how checkpoint kinases Mec1p/ATR and Rad53p/CHK2 regulate transcription termination. Because maintenance of genome stability is crucial for survival, cells have evolved highly conserved mechanisms to sense and signal damaged DNA. These mechanisms are collectively referred to as the DNA damage checkpoint (DDC). In addition to DDC, eukaryotic cells have DNA replication checkpoint (DRC) that specifically signals slowly progressing or arrested replication forks. A cascade of checkpoint kinases is the key component of both DDC and DRC. Activation of DDC or DRC triggers transcriptional reprogramming, required for coping with genotoxic or replicative stress. Why is the involvement of Mec1p and Rad53p in transcription termination important? During S phase, transcription and replication machineries compete for the same DNA template and can interfere with each other and cause DNA damage. Defects in transcription termination lead to increased levels of R loops and genome instability, indicating that rapid removal of transcription complexes at the ends of genes is important for reducing interference between transcription and replication. Our preliminary data show that replication stress triggers accumulation of RNA polymerase II (RNAPII) at the 3’ ends of genes. The RNAPII accumulation is exacerbated when the checkpoint kinase Mec1p is inactivated, suggesting Mec1p involvement in transcription termination. This notion is also supported by our data indicating a reduced cleavage of pre-mRNAs at the polyadenylation sites upon Mec1p or Rad53p inactivation. The central hypothesis of this proposal is that checkpoint kinases Mec1p and Rad53p regulate pre-mRNA 3’ end processing and transcription termination. This hypothesis will be tested in two Aims. In Aim 1, we will determine the mechanism of how Mec1p and Rad53p regulate the transcription termination by polyadenylation-dependent pathway. In Aim 2, we will determine the mechanism of how Mec1p and Rad53p regulate the transcription termination by Nrd1p-Nab3p-Sen1p (NNS)-dependent pathway. Experiments in both Aims will use yeast Saccharomyces cerevisiae as a model organism. Yeast is an ideal organism for the proposed studies, since it allows use of genetic, molecular, and biochemical tools, and is very suitable for the involvement of undergraduate students who will participate in all aspects of the study. Since dysregulation of transcription termination is associated with DNA damage, genome instability, and cancer, analysis of the regulatory links between DDC/DRC and transcription termination will contribute to the identification of novel targets and approaches for cancer treatment. This project is innovative because it reveals new and unexpected connection between checkpoint kinases Mec1p/ATR and Rad53p/CHK2 and termination of transcription. This project will also provide an excellent research environment for motivated undergraduate students.

项目摘要 本项目的目标是了解检查点激酶Mec 1 p/ATR和Rad 53 p/CHK 2如何调节 转录终止由于维持基因组的稳定性对生存至关重要， 高度保守的机制来感知和发送受损DNA的信号。这些机制共同 DNA损伤检查点（DDC）除了DDC，真核细胞还有DNA复制 检查点（DRC），专门发出缓慢进展或停滞的复制叉的信号。的级联 检查点激酶是DDC和DRC的关键组分。DDC或DRC触发器激活 转录重编程，需要应对遗传毒性或复制压力。为什么会牵扯到 Mec 1 p和Rad 53 p在转录终止中的重要性？在S期，转录和复制 机器竞争相同的DNA模板，并且可以相互干扰并导致DNA损伤。 转录终止缺陷导致R环水平增加和基因组不稳定性，表明 快速去除基因末端的转录复合物对于减少基因间的干扰是重要的。 转录和复制。我们的初步数据表明，复制应激触发RNA的积累， RNA聚合酶II（RNAPII）在基因的3'末端。RNAPII积累加剧时， 检查点激酶Mec 1 p失活，表明Mec 1 p参与转录终止。这 我们的数据也支持这一观点，表明前体mRNA在多聚腺苷酸化位点的切割减少 在Mec 1 p或Rad 53 p失活后。该建议的中心假设是，检查点激酶Mec 1 p Rad 53 p调控前体mRNA 3'端加工和转录终止。这一假设将是 测试了两个目标。在目标1中，我们将确定Mec 1 p和Rad 53 p如何调节细胞凋亡的机制。 通过多聚腺苷酸化依赖性途径终止转录。在目标2中，我们将确定机制 Mec 1 p和Rad 53 p如何调节Nrd 1 p-Nab 3 p-Sen 1 p（NNS）依赖的转录终止 通路这两个目标的实验将使用酵母酿酒酵母作为模式生物。酵母是 一个理想的生物体为拟议的研究，因为它允许使用遗传，分子和生物化学工具， 非常适合本科生的参与，他们将参与各个方面的工作， study.由于转录终止的失调与DNA损伤、基因组不稳定性、 和癌症，DDC/DRC和转录终止之间的调控联系的分析将有助于 用于癌症治疗的新靶点和方法的鉴定。该项目具有创新性，因为 它揭示了检查点激酶Mec 1 p/ATR和Rad 53 p/CHK 2之间新的和意想不到的联系， 转录终止。该项目还将提供一个良好的研究环境， 本科生。