Terminal DNA structure and ATP influence binding parameters of the DNA-dependent protein kinase at an early step prior to DNA synapsis.
The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates the non-homologous end-joining pathway of DNA double-strand break repair in mammalian cells. The ability of DNA-PKcs to sense and respond to different terminal DNA structures is postulated to be important for its regulatory function. It is unclear whether discrimination occurs at the time of formation of the initial protein-DNA complex or later, at the time of formation of a paired, or synaptic complex between opposing DNA ends. To gain further insight into the mechanism of regulation, we characterized the binding of DNA-PKcs to immobilized DNA fragments that cannot undergo synapsis. Results showed that DNA-PKcs strongly discriminates between different terminal structures at the time of initial complex formation. Although Ku protein stabilizes DNA-PKcs binding overall, it is not required for discrimination between terminal structures. Base mispairing, temperature and the presence of an interstrand linkage influence the stability of the initial complex in a manner that suggests a requirement for DNA unwinding, reminiscent of the 'open complex' model of RNA polymerase-promoter DNA interaction. ATP and a nonhydrolyzable ATP analog also influence the stability of the DNA-PKcs*DNA complex, apparently by an allosteric mechanism that does not require DNA-PKcs autophosphorylation.
Jovanovic
Marko
M
Program in Cancer Biology and Gene Regulation, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA.
Dynan
William S
WS
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D034961:Antigens, Nuclear / Q000378:metabolism; D023902:Chromosome Pairing; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D010766:Phosphorylation; D011485:Protein Binding; D020349:Surface Plasmon Resonance
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D051747:DNA-Activated Protein Kinase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen