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 该项目的目的是了解检查点激酶MEC1P/ATR和RAD53P/CHK2调节 转录终止。由于基因组稳定性的维持对于生存至关重要，因此细胞已经发展 高度保守的机制，以感知和信号受损的DNA。这些机制集体 称为DNA损伤检查点（DDC）。除DDC外，真核细胞具有DNA复制 CheckPoint（DRC）特别发出慢慢进行或逮捕复制叉的信号。一个级联 检查点激酶是DDC和DRC的关键组成部分。 DDC或DRC触发器的激活 转录重编程，以应对遗传毒性或复制应力所需。为什么参与 转录终止中的MEC1P和RAD53P很重要吗？在S阶段，转录和复制 机械竞争相同的DNA模板，可以互相干扰并造成DNA损伤。 转录终止缺陷导致R环和基因组不稳定性的水平增加，表明 快速去除基因末端的转录复合物对于减少干扰很重要 转录和复制。我们的初步数据表明，复制应力会触发RNA的积累 基因3'末端的聚合酶II（RNAPII）。当RNAPII积累会加剧 检查点激酶MEC1P被灭活，表明MEC1P参与转录终止。这 我们的数据还支持了概念，表明在聚腺苷酸位点降低了前MRNA的裂解 MEC1P或RAD53P灭活后。该提议的中心假设是检查点激酶MEC1P RAD53P调节前MRNA 3'末端处理和转录终止。这个假设将是 以两个目标进行测试。在AIM 1中，我们将确定MEC1P和RAD53P如何调节的机制 通过聚腺苷酸化途径的转录终止。在AIM 2中，我们将确定机制 MEC1P和RAD53P如何通过NRD1P-NAB3P-SEN1P（NNS）依赖性调节转录终止 路径。这两个目标的实验都将使用酿酒酵母作为模型生物。酵母是 拟议研究的理想有机体，因为它允许使用遗传，分子和生化工具， 并且非常适合将参与各个方面的本科生的参与 学习。由于转录终止的失调与DNA损伤，基因组不稳定性有关，因此 和癌症，对DDC/DRC与转录终止之间的调节联系的分析将有助于 鉴定新的癌症治疗目标和方法。这个项目是创新的，因为 它揭示了检查点激酶MEC1P/ATR和RAD53P/CHK2和 终止转录。该项目还将为融合提供出色的研究环境 本科生。