Correspondence to:

Cisplatin is widely used against various tumors, but resistance is commonly encountered. By inducing DNA crosslinks, cisplatin triggers DNA damage response (DDR) and cell death. However, the molecular determinants of how cells respond to cisplatin are incompletely understood. Since ubiquitination plays a major role in DDR, we performed a high-content siRNA screen targeting 327 human ubiquitin ligases and 92 deubiquitinating enzymes in U2OS cells, interrogating the response to cisplatin. We quantified γH2AX by immunofluorescence and image analysis as a read-out for DNA damage. Among known mediators of DDR, the screen identified the ubiquitin ligase G2E3 as a new player in the response to cisplatin. G2E3 depletion led to decreased γH2AX levels and decreased phosphorylation of the checkpoint kinase 1 (Chk1) upon cisplatin. Moreover, loss of G2E3 triggered apoptosis and decreased proliferation of cancer cells. Treating cells with the nucleoside analogue gemcitabine led to increased accumulation of single-stranded DNA upon G2E3 depletion, pointing to a defect in replication. Furthermore, we show that endogenous G2E3 levels in cancer cells were down-regulated upon chemotherapeutic treatment. Taken together, our results suggest that G2E3 is a molecular determinant of the DDR and cell survival, and that its loss sensitizes tumor cells towards DNA-damaging treatment