ABSTRACT
The DNA-dependent protein kinase (DNA-PK) complex plays a crucial role in radiation-induced DNA damage recognition. The complex includes the ku heterodimer, which comprises ku 70 and ku 80 subunits, that binds DNA termini of breaks without sequence specificity, and the catalytic subunit DNA-PKCS: The activation of the DNA-PK complex was studied in X-irradiated peripheral blood mononuclear cells (PBMC) from subjects of different ages. Radiation-induced changes in the DNA-binding activity of the ku heterodimer, and in the concentrations of ku 70, ku 80, DNA-PKcs and phosphorylated ku 80 were determined in nuclear and cytoplasmic extracts. DNA-binding activity was increased by irradiation only in the nuclear extract of PBMC from young, but not from elderly subjects, whereas it was found unchanged in cytoplasmic extracts regardless of age. The radiation-induced activation of the DNA-PK complex may result from the increased concentrations of ku 80 and DNA-PKcs in the cytoplasm of PBMC from young, but not from elderly subjects, leading to a higher concentration of phosphorylated ku 80 which readily migrates to the nucleus where, after dimerization with ku 70, binds to DNA breaks. These findings suggest major steps involved in DNA-PK activation, and the intracellular and molecular changes that may account for the age-dependent impairment of DNA repair capacity in irradiated mammalian cells.
Subject(s)
Antigens, Nuclear , DNA Damage , DNA Helicases , Leukocytes, Mononuclear/enzymology , Leukocytes, Mononuclear/radiation effects , Protein Serine-Threonine Kinases/physiology , Adult , Age Factors , Aged , Aged, 80 and over , Cell Nucleus/enzymology , Cell Nucleus/metabolism , Cell Nucleus/radiation effects , Cytoplasm/enzymology , Cytoplasm/metabolism , Cytoplasm/radiation effects , DNA/metabolism , DNA/radiation effects , DNA-Activated Protein Kinase , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/radiation effects , Dimerization , Enzyme Activation/radiation effects , Humans , Ku Autoantigen , Macromolecular Substances , Male , Molecular Weight , Nuclear Proteins/metabolism , Nuclear Proteins/radiation effects , Phosphorylation/radiation effects , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/radiation effectsABSTRACT
We have investigated the effects of an interleukin (IL)-6-type cytokine on the DNA-binding activity of ku and on unscheduled DNA repair in X-ray-treated peripheral blood mononuclear cells (PBMC) from human subjects of different ages. The cytokine used, called K-7/D-6, is an IL-6 variant with increased in vivo and in vitro biological activity compared to the wild type molecule. Ku is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK). It binds the ends of various types of DNA discontinuity and is involved in the repair of DNA breaks caused by V(D)J recombination, isotype switching, physiological oxidation reactions, ionizing radiation and some chemotherapeutic drugs. The ku-dependent repair process, called non-homologous end joining, is the main DNA double strand break repair mechanism in irradiated mammalian cells. Results show that K-7/D-6 significantly increases DNA-binding activity of ku in irradiated PBMC from young but not from elderly subjects. However, K-7/D-6 is able to induce unscheduled DNA repair in irradiated PBMC from both young and elderly subjects. These effects of K-7/D-6 are relevant to the mechanisms of the cellular response to DNA damage.
