Identification of RNF8 as a ubiquitin ligase involved in targeting the p12 subunit of DNA polymerase δ for degradation in response to DNA damage.

DNA polymerase delta consists of four subunits, one of which, p12, is degraded in response to DNA damage through the ubiquitin-proteasome pathway. However, the identities of the ubiquitin ligase(s) which are responsible for the proximal biochemical events in triggering proteasomal degradation of p12 are unknown. We employed a classical approach to identifying a ubiquitin ligase that is involved in p12 degradation. Using UbcH5c as ubiquitin conjugating enzyme, a ubiquitin ligase activity that polyubiquitinates p12 was purified from HeLa cells. Proteomic analysis revealed that RNF8, a RING finger ubiquitin ligase that plays an important role in the DNA Damage Response, was the only ubiquitin ligase present in the purified preparation. In vivo, DNA damage induced p12 degradation was significantly reduced by shRNA knockdown of RNF8 in cultured human cells and in RNF8-/- mouse epithelial cells. These studies provide the first identification of a ubiquitin ligase activity that is involved in the DNA damage induced destruction of p12. The identification of RNF8 allows new insights into the integration of the control of p12 degradation by different DNA damage signaling pathways.