DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus.
Double-strand breaks (DSBs) can be efficiently removed from the DNA of higher eukaryotes by nonhomologous end-joining (NHEJ). Genetic studies implicate the DNA-dependent protein kinase (DNA-PK) in NHEJ, but the exact function of this protein complex in the rejoining reaction remains to be elucidated. We compared rejoining of DNA DSBs in a human glioma cell line, M059-J, lacking the catalytic subunit of DNA-PK (DNA-PKcs), and their isogenic but DNA-PK-proficient counterpart, M059-K. In both cell lines, rejoining of DNA DSBs was biphasic, with a fast and a slow component operating with a half-life of approximately 22 min and 12 h, respectively. Deficiency in DNA-PK activity did not alter the half-times of either of these components of rejoining but increased from 17 to 72% the proportion of DNA DSB rejoining with slow kinetics. DNA DSB rejoining was nearly complete in both cell lines, and there was only a small increase in the number of unrejoined breaks in M059-J as compared with M059-K cells after 30 h of incubation. Wortmannin radiosensitized to killing M059-K cells and strongly inhibited DNA DSB rejoining. Wortmannin did not affect the radiosensitivity to killing and produced only a modest inhibition in DNA DSB rejoining in M059-J cells, suggesting that, for these end points, DNA-PK is the principal target of the drug. These observations demonstrate that DNA-PK deficiency profoundly decreases the proportion of DNA DSB rejoining with fast kinetics but has only a small effect on the fraction remaining unrejoined. We propose that in higher eukaryotes, an evolutionarily conserved, independently active, but inherently slow NHEJ pathway is stimulated 30-fold by DNA-PKcs to rapidly remove DNA DSBs from the genome. The stimulation is expected to be of local nature and the presence of DNA-PKcs in the vicinity of the DNA DSB determines whether rejoining will follow fast or slow kinetics. Structural and regulatory functions of DNA-PKcs may mediate this impressive acceleration of DNA DSB rejoining, and regions of chromatin within a certain range from this large protein may benefit from these activities. We propose the term DNA-PK surveillance domains to describe these regions.
DiBiase
S J
SJ
Department of Radiation Oncology, Kimmel Cancer Center, Philadelphia, Pennsylvania 19107, USA.
Zeng
Z C
ZC
Chen
R
R
Hyslop
T
T
Curran
W J
WJ
Iliakis
G
G
D001932:Brain Neoplasms / Q000235:genetics*; D004249:DNA Damage; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000235:genetics*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D005910:Glioma / Q000235:genetics*; D006801:Humans; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases