Novel mechanisms in activation of ATR kinase

Radiotherapy and many chemotherapeutic drugs work by inducing lethal DNA damage in cancer cells. However, networks of signaling cascades that are induced upon DNA damage may counteract this by leading to repair of the damaged DNA. The ATR kinase is a key component of these signaling networks, and inhibitors of ATR are consequently under evaluation for combination treatment with chemo- or radiotherapy. DNA damage can also arise due to errors during normal cell growth. Such errors frequently occur when cells are replicating their DNA, due to conflicts with other processes occurring on DNA such as transcription. At the start of this project, we had recently discovered a new mechanism of ATR activation via the transcription machinery. The main goals of the project were to explore this mechanism further and to understand how it may affect DNA replication. Our project results suggest that ATR is activated due to signaling from RNA polymerase 2, the main enzyme catalyzing transcription of DNA into mRNA. The phosphatase PNUTS-PP1 suppresses ATR activity by dephosphorylating RNA polymerase II. Furthermore, CDC73, a factor which binds to the phosphorylated RNA polymerase II, contributes to regulate ATR activity. In addition, we have found that PNUTS-PP1 together with its binding partner WDR82 are required for normal DNA replication, as they contribute to prevent conflicts between replication and transcription. Particularly we have found that proper dephosphorylation of RNA polymerase 2 is required for its degradation and timely removal from chromatin. If the dephosphorylation is inhibited, RNA polymerase 2 is less degraded and is therefore present for abnormally long times on chromatin during transcription. This leads to more collisions between the transcription and replication machineries in the cells, resulting in severe hindering of DNA replication. During this work we have also developed a new method to measure chromatin bound RNA polymerase 2 in single cells, by use of flow cytometry. This method includes analysis of different phosphorylated forms of RNA polymerase 2, which represent different steps of the transcription process. Using this method we discovered that promoter proximal RNA polymerase 2 is degraded. Altogether, our results provide new knowledge about non-canonical pathways of ATR activation and of factors involved in preventing conflicts between DNA transcription and DNA replication.

Resultatene har gitt økt kunnskap om DNA-skade-signalveier. Særlig har resultatene bidratt til økt forståelse av hvordan ATR-kinasen aktiveres. Resultatene har også gitt økt forståelse av hvordan samspillet mellom prosessene transkripsjon og replikasjon er regulert i cellene, for å unngå replikasjonsstress og påfølgende DNA-skade. I tillegg har resultatene bidratt til økt kunnskap om hvordan transkripsjon påvirkes av DNA-skade og hvordan reparasjon av dobbelttrådbrudd påvirkes av transkripsjonsprosessen. På kort sikt forventes disse resultatene særlig å ha akademisk nytteverdi for andre forskere i fagfeltet, slik at de bidrar til at det genereres ny kunnskap. På lang sikt forventes resultatene å kunne ha nytteverdi for å utvikle nye måter å forbedre kreftbehandling med f.eks. stråleterapi, hvor kreftcellene drepes ved induksjon av DNA-skade. En PhD-stipendiat har vært ansatt på prosjektet og disputas er planlagt høsten 2023.

In response to DNA damage or replication stress human cells activate a network of signaling cascades to promote cell survival. The ATR kinase is a key component of this signaling network, being a major orchestrator of DNA damage repair and cell cycle checkpoint pathways. Understanding how ATR is activated is therefore a key issue in the DNA damage field. In this project we will address a novel hypothesis for how ATR is activated in human cells. We hypothesize that a major signal promoting ATR activation comes from the transcription machinery. The new hypothesis is based on our own present work with the Protein Phosphatase 1 (PP1) nuclear targeting factor Pnuts, which mediates dephosphorylation of RNA polymerase II. Our preliminary results strongly suggest that phosphorylated RNA pol2 provides a signal to activate ATR. To address our new hypothesis, we will apply phosphorylation defective mutants of RNA pol2, and utilize both a hypothesis-driven approach and unbiased proteomics techniques to elucidate the signaling cascade from phosphorylated RNA pol2 towards ATR. In addition, we will address whether the phosphorylated RNA pol2 also afects DNA replication dynamics. The latter may help to identifying novel mechanisms underlying conflicts between the transcription and replication machnieries in human cells. Our results will reveal novel knowledge about non-canonical pathways of ATR activation. Since ATR plays a major role in maintaining genome integrity and ATR-inhibitors are in progress for potential cancer treatment, such knowledge will be highly important.