Abstract
The DNA-dependent protein kinase (DNA-PK) controls the repair of double-stranded DNA breaks in mammalian cells. The protein kinase subunit of DNA-PK (DNA-PKcs) is targeted to DNA breaks by association with the Ku DNA-binding heterodimer. Here we show that a Ku association site is present at the carboxyl terminus of DNA-PKcs (amino acids 3002-3850) near the protein kinase domain. Correspondingly, the nuclear c-Abl tyrosine kinase that associates with DNA-PK also binds to the kinase homology domain. The c-Abl SH3 domain binds to amino acids 3414-3850 of DNA-PKcs. c-Abl phosphorylates C-terminal fragments of DNA-PKcs, particularly amino acids 3414-3850. c-Abl phosphorylation of DNA-PKcs disassociates the DNA-PKcs.Ku complex. Thus, Ku and c-Abl provide opposing functions with regard to DNA-PK activity.
Highlights
The DNA-dependent protein kinase (DNA-PK) controls the repair of double-stranded DNA breaks in mammalian cells
DNA double-strand break (DSB)1 repair occurs in eukaryotic cells following the formation of chromosome breaks by spontaneous damage or in V(D)J gene rearrangement of lymphoid cell differentiation
Much of the same repair machinery is necessary for cell survival following ionizing radiation (IR) damage, known to include DSB repair [1, 2]
Summary
The DNA-dependent protein kinase (DNA-PK) controls the repair of double-stranded DNA breaks in mammalian cells. The pools of Ku and DNA-PKcs are not always associated in eukaryotic cells, allowing activation and alternative regulation of the kinase by effector protein binding. Aliquots of 293 lysates (0.5–1.0 mg) were mixed with equal amounts of each [35S]DNA-PKcs in vitro translation product (10 –20 l), diluted to reduce the NaCl concentration to 150 –170 mM (final volume ϭ 350 – 450 l), and incubated on ice for 1–3 h.
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