Understanding the differential consequences ofcheckpoint adaptation following DNA damagein repair-proficient and repair-deficient cells

Abstract

When cells experience DNA damage, they halt the cell cycle before sister chromatid separation has begun in response to the activation of the DNA damage checkpoint. This surveillance mechanism provides time to repair the damage and only when repair has been successful the cell cycle is resumed. Therefore, cell cycle arrest and damage repair are important processes to ensure the stability of the genome and the faithful transfer of genetic information to daughter cells. However, if repair is not possible, cells can override the DNA damage checkpoint and terminate the cell cycle arrest by a mechanism called (checkpoint) adaptation. Although many proteins have been shown to be involved in the adaptation process, its molecular mechanisms still remain elusive. Especially the critical determinants initiating checkpoint adaptation have not been fully identified. Understanding this pathway is of particular interest since checkpoint adaptation is a driving force of genome instability and has detrimental consequences including cell death and various genomic aberrations. Interestingly, the concept of checkpoint adaptation is not only found in unicellular eukaryotes like yeast but also in multicellular organisms. Especially during cancerogenesis, checkpoint adaptation is thought to contribute to genome instability. We could previously show that inhibition of the highly conserved TOR nutrient signalling pathway either by genetic or pharmacologic means prevents checkpoint adaptation in Saccharomyces cerevisiae. These observations suggests that nutrient signalling pathways involving TOR signalling node play an important role in response to DNA damage. We set out to further investigate the link between nutrient signalling, checkpoint adaptation and genome stability. We show that prevention of adaptation can be achieved by modulating the Tap42-PP2A axis downstream of TOR signalling. We found that pharmacological inhibition of TOR by rapamycin affects protein levels of Cdc5, a major factor promoting adaptation. Using RAD52-deficient yeast cells to mimic the DNA repair defect observed in many human cancers, we confirmed previous results showing that preventing adaptation sensitizes these cells to genotoxins. However, if adaptation is allowed to occur, repair-deficient cells acquire genotoxin resistance and display an aneuploid karyotype. Gene expression profiling revealed that resistant repair-defective yeast cells exhibit common aneuploidy-associated phenotypes. Although resistant aneuploid cells are still checkpoint-competent, they can proliferate in the presence of persistent DNA damage. This underlines the role of checkpoint adaptation in the acquisition of genotoxin resistance. Taken together, our results highlight an intriguing relationship between the DNA damage response and genome stability, which appears to be associated with checkpoint adaptation and nutrient signalling pathways. Furthermore, using an easily tractable model organism such as budding yeast, we provide insights into the relationship between fundamental and highly conserved cellular processes that could be useful for drug development and disease treatment in humans as well.