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Abstract
Nonhomologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand
breaks (DSBs) in higher eukaryotes. Several proteins, including the DNA-dependent
protein kinase (DNA-PK), XRCC4 and DNA ligase IV, are required for nonhomologous end
joining both in vitro and in vivo. Since XRCC4 is recruited to the DNA double-strand
break with DNA-PK, and because the protein kinase activity of DNA-PK is required for
its in vivo function, we reasoned that XRCC4 could be a potential physiological substrate
of DNA-PK. Here, we have used mass spectrometry to map the DNA-PK phosphorylation
sites in XRCC4. Two major phosphorylation sites (serines 260 and 318), as well as
several minor sites were identified. All of the identified sites lie within the carboxy-terminal
100 amino acids of XRCC4. Substitution of each of these sites to alanine (in combination)
reduced the ability of DNA-PK to phosphorylate XRCC4 in vitro by at least two orders
of magnitude. However, XRCC4-deficient cells that were complemented with XRCC4 lacking
DNA-PK phosphorylation sites were analogous to wild type XRCC4 with respect to survival
after ionizing radiation and ability to repair DSBs introduced during V(D)J recombination.