Abstract
Non-homologous end-joining is the primary repair pathway for DNA double-strand breaks in human cells. The rejoining of DNA ends is catalyzed by the complex of DNA ligase IV and XRCC4. Using human XRCC4-knockout cells that have no detectable yield of Ligase IV, we analyzed the end-joining (EJ) activity in their extracts. As a result, the activity in XRCC4-knockout cells is lower than that in the parental cells with XRCC4 but is still high under our in vitro conditions. The XRCC4-independent EJ activity was partially purified through chromatographic procedures, leading into the separation from DNA-PK components, DNA-PKcs and Ku70/Ku86. Interestingly, the addition of DNA-PK components to the active fraction had no effect on its EJ activity, while they completely suppressed the activity of T4 phage-derived DNA ligase. Also, in combination with DNA-PK, the coexistence of a kinase inhibitor wortmannin greatly abolished the EJ activity. These results suggest that the XRCC4-independent EJ is associated with the function of DNA-PK complex, whose kinase activity is a regulatory factor for the EJ.
