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DNA Repair (Amst). 2003 Nov 21;2(11):1239-52.

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

Author information

1
Department of Biochemistry & Molecular Biology, University of Calgary, 3330 Hospital Drive, NW, Calgary, Alta, Canada T2N 1N4.

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.

PMID:
14599745
[Indexed for MEDLINE]

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