Abstract
DNA-dependent protein kinase (DNA-PK) is considered the "sensor" of DNA double-strand breaks (DSBs) and thought to play an essential role in DSB repair and recombination through non-homologous end-joining (NHEJ) pathway. It is shown that the protein kinase activity of DNA-PK is required for its function and that DNA-PK is capable of phosphorylating a number of proteins in vitro. However, the substrate(s) in vivo and the significance of phosphorylation have remained to be clarified for many years.
We have investigated the phosphorylation by DNA-PK of XRCC4, which is thought to join two DNA ends in cooperation with DNA ligase IV. We have so far identified four phosphorylation sites, in addition to two identified by others. Two of the above phosphorylation sites are really phosphorylated in living cells after irradiation in a manner dependent on DNA-PKcs. Further, the mutant lacking these phosphorylation sites exhibited elevated radiosensitivity with reduced DNA repair capability. These results indicated that XRCC4 is one of, at least, the true substrates of DNA-PK in vivo and the phosphorylation is really important in DSB repair. We will also discuss the role of phosphorylation in the assembly/disassembly of the DSB repair machinery based on the analysis of XRCC4-chromatin binding status through biochemical fractionation.
Furthermore, we would summarize the current understanding and discuss future directions of this issue.
