UC Riverside

DNA double-strand breaks (DSBs) can be induced endogenously and by exposure to ionizing radiation, and they represent one of the most deleterious forms of DNA damage. To avoid genomic stability arising from DNA DSBs, cells employ DNA damage response (DDR) pathways to detect and promptly repair DNA DSBs. In mammalian cells, the DDR pathway involves a cascade of events of protein recruitments and chromatin modifications near the damaged regions of DNA. An important chromatin modification known to occur during this process is the ubiquitination of histone H2A and H2AX by the E3 ubiquitin ligase RNF168. Herein, we identified, for the first time, the phosphorylation of serine 97 in RNF168, and we found that the level of this phosphorylation was significantly increased in cultured human cells upon exposure to γ rays or a radiomimetic agent, neocarzinostatin. Additionally, the introduction of S97A or S97D mutation to RNF168 substantially reduced the protein’s ubiquitination activity in cells, diminished the efficiency of DNA DSB repair via the non-homologous end-joining pathway, and rendered cells more sensitive toward ionizing radiation. Together, phosphorylation of S97 in RNF168 plays a significant role in the cellular tolerance and repair of DNA DSBs. Thus, our study provided important insights into the post-translational regulation of RNF168 in DNA damage response.