Regulation of transcription termination by checkpoint kinases Mec1p and Rad53p

St. John'S University, Queens NY

Investigators

Abstract

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.