Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks.
The DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PK(CS) recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PK(CS) accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PK(CS) influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PK(CS) at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PK(CS) influence the stability of its binding to DNA ends. We suggest a model in which DNA-PK(CS) phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PK(CS) with the DNA ends.
Uematsu
Naoya
N
Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Weterings
Eric
E
Yano
Ken-ichi
K
Morotomi-Yano
Keiko
K
Jakob
Burkhard
B
Taucher-Scholz
Gisela
G
Mari
Pierre-Olivier
PO
van Gent
Dik C
DC
Chen
Benjamin P C
BP
Chen
David J
DJ
D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D016466:CHO Cells; D020134:Catalytic Domain; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D007834:Lasers; D010766:Phosphorylation; D038761:Photobleaching
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen