A mechanism for DNA-PK activation requiring unique contributions from each strand of a DNA terminus and implications for microhomology-mediated nonhomologous DNA end joining.
DNA-dependent protein kinase (DNA-PK) is an essential component of the nonhomologous end joining pathway (NHEJ), responsible for the repair of DNA double-strand breaks. Ku binds a DSB and recruits the catalytic subunit, DNA-PKcs, where it is activated once the kinase is bound to the DSB. The precise mechanism by which DNA activates DNA-PK remains unknown. We have investigated the effect of DNA structure on DNA-PK activation and results demonstrate that in Ku-dependent DNA-PKcs reactions, DNA-PK activation with DNA effectors containing two unannealed ends was identical to activation observed with fully duplex DNA effectors of the same length. The presence of a 6-base single-stranded extension resulted in decreased activation compared to the fully duplex DNA. DNA-PK activation using DNA effectors with compatible termini displayed increased activity compared to effectors with noncompatible termini. A strand orientation preference was observed in these reactions and suggests a model where the 3' strand of the terminus is responsible for annealing and the 5' strand is involved in activation of DNA-PK. These results demonstrate the influence of DNA structure and orientation on DNA-PK activation and provide a molecular mechanism of activation resulting from compatible termini, an essential step in microhomology-mediated NHEJ.
Department of Biochemistry and Molecular Biology and Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46220, USA.
Katherine S
KS
Pawelczak
John J
JJ
Turchi
D004247:DNA / Q000737:chemistry*; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004789:Enzyme Activation; D008954:Models, Biological; D010766:Phosphorylation; D012689:Sequence Homology, Nucleic Acid
D004247:DNA; D051747:DNA-Activated Protein Kinase