DNA-PK phosphorylation sites in XRCC4 are not required for survival after radiation or for V(D)J recombination

• Published in: DNA repair Vol. 2; no. 11; pp. 1239 - 1252
• Main Authors: Yu, Yaping, Wang, Wei, Ding, Qi, Ye, Ruiqiong, Chen, Dawn, Merkle, Dennis, Schriemer, David, Meek, Katheryn, Lees-Miller, Susan P
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 21.11.2003; Elsevier
• Subjects: Alanine - metabolism; Amino Acid Sequence; Amino Acid Substitution; Bacteriology; Binding Sites; Biological and medical sciences; Cell Survival - radiation effects; DNA Damage; DNA double-strand break repair; DNA-Activated Protein Kinase; DNA-Binding Proteins - chemistry; DNA-dependent protein kinase; Dose-Response Relationship, Radiation; Fundamental and applied biological sciences. Psychology; Growth, nutrition, cell differenciation; HeLa Cells; Humans; Microbiology; Molecular and cellular biology; Molecular genetics; Mutagenesis. Repair; Nonhomologous end joining; Nuclear Proteins; Phosphorylation; Protein-Serine-Threonine Kinases - metabolism; Radiation, Ionizing; Recombination, Genetic; V(D)J recombination; VDJ Recombinases - metabolism; XRCC4; XRCC4 protein; DNA-PKcs; DNA double-strand break repair; Phosphorylation; DNA-PK; hXRCC4; MALDI-TOF; XRCC4; DTT; V(D)J recombination; Nonhomologous end joining; TFA; DSB; mXRCC4; DNA-dependent protein kinase; NHEJ; HPLC; SDS PAGE; Recombination; Enzyme; Transferases; Joining; Survival; Double strand break; Double stranded DNA; Protein kinase; End; Repair
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/S1568-7864(03)00143-5

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.