Abstract
The nonhomologous end-joining (NHEJ) pathway is conserved in eukaryotes, from yeast to humans. Without requiring homologous DNA, NHEJ repairs DNA double-strand breaks produced by xenobiotic agents, such as topoisomerase II inhibitors and ionizing radiation, or by the cellular pathway for V(D)J recombination of the immunoglobulin genes. The key proteins required for NHEJ include the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), the Ku70/80 heterodimer, the XRCC4-DNA ligase IV complex and recently found molecule XLF/Cernunnos. From all of these key proteins, DNA-PKcs plays an important role in mediating the repair process through NHEJ repair. Moreover, the protein kinase activity of DNA-PK is required for its in vivo function, as DNA-PKcs containing inactivating mutations in the catalytic domain does not complement the radiosensitive phenotype of DNA-PKcs-deficient cells. DNA-PKcs is known to phosphorylates tens of proteins in vitro but the true substrate in vivo and the significance of phosphorylation in NHEJ remains to be clarified. So, there is need to identify the genuine phosphorylation targets of DNA-PK and found physiological significance of these phosphorylation targets. In the present investigations we have identified several new phosphorylation sites in XLF and XRCC4 protein by DNA-PK in vitro. We have prepared phosphorylation-specific antibodies to respective sites and observed that some of these phosphorylation sites were indeed phosphorylated in the living cells following irradiation. To explore the biological significance of phosphorylation, we established cells expressing phosphorylation-defective mutants.
