Identification of DNA-PKcs phosphorylation sites in XRCC4 and effects of mutations at these sites on DNA end joining in a cell-free system.
Nonhomologous end joining (NHEJ) is the principal mechanism for repairing DNA double-strand breaks in mammalian cells. NHEJ requires at least three protein components: the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku protein, and the DNA ligase IV/XRCC4 (DNL IV/XRCC4) complex. Although DNA-PKcs phosphorylates several sites within itself and these other proteins, the significance of phosphorylation at individual sites is not yet understood. Here we investigate the effects of DNA-PKcs-mediated phosphorylation at two sites in XRCC4. One is a previously described site at serine 260; the other is a newly mapped site at serine 318. XRCC4 bearing mutations at these sites was co-expressed with DNL IV, the resulting complexes were purified, and activity was tested in a cell-free end-joining system reconstituted from recombinant and purified proteins. Substitution of alanine for serine 260 or 318, which prevents phosphorylation at these positions, or aspartate for serine 260, which mimics constitutive phosphorylation, had no significant effect on overall end-joining activity. In the assay system used, DNA-PKcs is not essential, but when present, arrests the reaction until phosphorylation occurs, in effect establishing a reaction checkpoint. Mutations at serines 260 and 318 did not affect establishment or release from the checkpoint. Results demonstrate that DNA-PKcs-mediated phosphorylation of XRCC4 serine 260 and serine 318 does not directly control end-joining under the conditions tested.
Lee
Kyung-Jong
KJ
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Room CB-2803, Augusta, GA 30912, USA.
Jovanovic
Marko
M
Udayakumar
Durga
D
Bladen
Catherine L
CL
Dynan
William S
WS
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D016367:Baculoviridae; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D005982:Glutathione Transferase; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C101467:XRCC4 protein, human; D004247:DNA; D005982:Glutathione Transferase