Abstract
Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) in mammalian species. Using live cell imaging techniques, we have analyzed DSB responses of NHEJ core factors, including DNA-PKcs, XRCC4 and XLF. Based on our previous findings, we have proposed a new model of NHEJ assembly on DSBs, which comprises two critical phases. In the first phase, after the binding of Ku to DSBs, DNA-PKcs, XRCC4/LigIV, and XLF are recruited independently and simultaneously to DSBs. Subsequently, the recruited factors cooperatively interact with each other to form functional NHEJ machinery. In addition, we have demonstrated that Ku-XLF interaction occurs on DSBs, strongly indicating the critical role of the Ku-XLF interaction in the assembly of NHEJ machinery on DSBs. In this study, we performed a detailed analysis of the Ku-XLF interaction. First, we identified a domain in Ku heterodimer that is required for XLF interaction by a gel shift assay using purified Ku and XLF proteins. We confirmed that this domain is required for DSB response of XLF in vivo. Next, we identified an essential region of XLF for DSB accumulation in vivo. We expressed a mutant XLF lacking this region and found that the mutant XLF failed to associate with endogenous Ku. Interestingly, we observed significantly reduced association of the mutant XLF with endogenous XRCC4, although XRCC4 was previously reported to bind to a region distinct from the Ku-interacting region in vitro. These observations indicate that Ku-XLF interaction plays a critical role in cooperative interactions among the NHEJ core factors on DSBs, further supporting our model for DSB recognition in the NHEJ pathway.
