RNF8 and RNF168 but not HERC2 are required for DNA damage-induced ubiquitylation in chicken DT40 cells

The ubiquitylation cascade plays an important role in the recruitment of repair factors at DNA double-strand breaks. The involvement of a growing number of ubiquitin E3 ligases adds to the complexity of the DNA damage-induced ubiquitin signaling. Here we use the genetically tractable avian cell line DT40 to investigate the role of HERC2, RNF8 and RNF168 in the DNA damage-induced ubiquitylation pathway. We show that formation of ubiquitin foci as well as cell survival after DNA damage depends on both RNF8 and RNF168. However, we find that RNF8 and RNF168 knockout cell lines respond differently to treatment with camptothecin indicating that they do not function in a strictly linear manner. Surprisingly, we show that HERC2 is required neither for survival nor for ubiquitin foci formation after DNA damage in DT40. Moreover, the E3 ubiquitin ligase activity of HERC2 is not redundant to that of RNF8 or RNF168.

► We have constructed HERC2−/−, RNF8−/− and RNF168−/− DT40 cell lines.
► RNF8 and RNF168 but not HERC2 are needed to form ubiquitin foci after DNA damage.
► RNF8−/− and RNF168−/− but not HERC2−/− cell lines are sensitive to X-rays and CPT.
► Epistasis analysis of RNF8−/− and RNF168−/− cell lines reveals a non-linear pathway.