The Ku80 carboxy terminus stimulates joining and artemis-mediated processing of DNA ends.
Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) and the XRCC4/ligase IV complex. Activation of the DNA-PK(CS) serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PK(CS) at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PK(CS) autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PK(CS) autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.
Weterings
Eric
E
Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9187, USA.
Verkaik
Nicole S
NS
Keijzers
Guido
G
Florea
Bogdan I
BI
Wang
Shih-Ya
SY
Ortega
Laura G
LG
Uematsu
Naoya
N
Chen
David J
DJ
van Gent
Dik C
DC
D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000502:physiology*; D020134:Catalytic Domain; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004720:Endonucleases; D006801:Humans; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments; D010766:Phosphorylation; D011839:Radiation, Ionizing; D017384:Sequence Deletion
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C468131:Ku80 antigen C-terminal peptide (720-732); D009687:Nuclear Proteins; D010446:Peptide Fragments; C101467:XRCC4 protein, human; D051747:DNA-Activated Protein Kinase; C426344:DCLRE1C protein, human; D004720:Endonucleases; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D045643:DNA Repair Enzymes