Regulation of transcription termination by checkpoint kinases Mec1p and Rad53p

项目总结 这个项目的目标是了解检查点激酶Mec1p/ATR和Rad53p/Chk2是如何调节的 转录终止。由于维持基因组的稳定性对生存至关重要，细胞已经进化 高度保守的机制，以感知和信号受损的DNA。这些机制统称为 称为DNA损伤检查点(DDC)。除了DDC，真核细胞还有DNA复制 检查点(DRC)，专门表示复制分叉进展缓慢或停止。一连串的 检查点激酶是DDC和DRC的关键组成部分。激活DDC或DRC触发器 转录重编程，是应对基因毒性或复制应激所必需的。为什么会有牵连 Mec1p和Rad53p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，可能会相互干扰，导致DNA损伤。 转录终止缺陷会导致R环水平增加和基因组不稳定，这表明 快速移除基因末端的转录复合体对于减少基因之间的干扰很重要 转录和复制。我们的初步数据显示，复制压力会触发RNA的积累 基因3‘端的聚合酶II(RNAPII)。当RNAPII的积累在 检查点激酶Mec1p失活，提示Mec1p参与转录终止。这 我们的数据也支持这一观点，即多聚腺苷化位点上的前-mRNAs切割减少 在Mec1p或Rad53p失活时。这一提议的中心假设是检查点激活Mec1p RAD53P调控Pre-mRNA3‘末端加工和转录终止。这一假设将是 在两个目标上进行了测试。在目标1中，我们将确定Mec1p和Rad53p如何调节 通过依赖于多聚腺苷的途径终止转录。在目标2中，我们将确定机制 Mec1p和Rad53p如何调节Nrd1p-NaB3p-Sen1p(NNS)依赖的转录终止 路径。这两个目标的实验都将使用酿酒酵母作为模式生物。酵母是 对于拟议的研究来说，这是一个理想的有机体，因为它允许使用遗传、分子和生化工具 并且非常适合本科生的参与，他们将参与到 学习。由于转录终止的失调与DNA损伤、基因组不稳定、 和癌症，分析DDC/DRC和转录终止之间的调控联系将有助于 用于确定癌症治疗的新靶点和新方法。这个项目具有创新性，因为 它揭示了检查点激酶Mec1p/ATR和Rad53p/Chk2之间新的和意想不到的联系，以及 终止转录。该项目还将为动力公司提供一个良好的研究环境 本科生。

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.