title: The kinase activity of DNA-PK is required to protect mammalian telomeres.
abstract: The kinase activity of DNA-dependent protein kinase (DNA-PK) is required for efficient repair of DNA double-strand breaks (DSB) by non-homologous end joining (NHEJ). DNA-PK also participates in protection of mammalian telomeres, the natural ends of chromosomes. Here we investigate whether the kinase activity of DNA-PK is similarly required for effective telomere protection. DNA-PK proficient mouse cells were exposed to a highly specific inhibitor of DNA-PK phosphorylation designated IC86621. Chromosomal end-to-end fusions were induced in a concentration-dependent manner, demonstrating that the telomere end-protection role of DNA-PK requires its kinase activity. These fusions were uniformly chromatid-type, consistent with a role for DNA-PK in capping telomeres after DNA replication. Additionally, fusions involved exclusively telomeres produced via leading-strand DNA synthesis. Unexpectedly, the rate of telomeric fusions induced by IC86621 exceeded that which occurs spontaneously in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) mutant cells by up to 110-fold. One explanation, that IC86621 might inhibit other, as yet unknown proteins, was ruled out when the drug failed to induce fusions in DNA-PKcs knock-out mouse cells. IC86621 did not induce fusions in Ku70 knock-out cells suggesting the drug requires the holoenzyme to be effective. ATM also is required for effective chromosome end protection. IC86621 increased fusions in ATM knock-out cells suggesting DNA-PK and ATM act in different telomere pathways. These results indicate that the kinase activity of DNA-PK is crucial to reestablishing a protective terminal structure, specifically on telomeres replicated by leading-strand DNA synthesis.
authors.affiliation: Department of Environmental and Radiological Health Sciences, Colorado State Univeristy, Fort Collins 80523, USA.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Bailey
authors.forename: Mark A
authors.initials: MA
authors.lastname: Brenneman
authors.forename: James
authors.initials: J
authors.lastname: Halbrook
authors.forename: Jac A
authors.initials: JA
authors.lastname: Nickoloff
authors.forename: Robert L
authors.initials: RL
authors.lastname: Ullrich
authors.forename: Edwin H
authors.initials: EH
authors.lastname: Goodwin
mesh_terms: D000098:Acetophenones / Q000378:metabolism*; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018929:Cell Culture Techniques; D018797:Cell Cycle Proteins; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009025:Morpholines / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016615:Telomere / Q000235:genetics*; D025521:Tumor Suppressor Proteins
chemical_list: C487214:1-(2-hydroxy-4-morpholin-4-ylphenyl)ethanone; D000098:Acetophenones; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009025:Morpholines; D025521:Tumor Suppressor Proteins; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases