Loss of Diphthamide Increases DNA Replication Stress in Mammalian Cells by Modulating the Translation of RRM1

Diphthamide (DPH) is a highly conserved post-translational modification exclusively present in eukaryotic translation elongation factor 2 (eEF2), with its loss leading to embryonic lethality in mice and developmental disorders in humans. In this study, we unveil the role of diphthamide in mammalian cell DNA damage stress, with a particular emphasis on DNA replication stress. We developed a systematic strategy to identify human proteins affected by diphthamide with a combination of computational profiling and quantitative proteomics. Through this approach, we determine that the translation of RRM1 is modulated by diphthamide via −1 frameshifting. Importantly, our results reveal that the dysregulation of RRM1 translation in DPH-deficient cells is causally linked to elevated DNA replication stress. These findings provide a potential explanation for how diphthamide deficiency leads to cancer and developmental defects in humans.