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The Journal of Neuroscience, June 15, 1999, 19(12):4727-4738

Changes in Expression of the DNA Repair Protein Complex DNA-Dependent Protein Kinase after Ischemia and Reperfusion

Deborah A. Shackelford, Takaaki Tobaru, Shengjia Zhang, and Justin A. Zivin

Department of Neurosciences, University of California at San Diego, La Jolla, California 92093-0624

Reperfusion of ischemic tissue causes an immediate increase in DNA damage, including base lesions and strand breaks. Damage is reversible in surviving regions indicating that repair mechanisms are operable. DNA strand breaks are repaired by nonhomologous end joining in mammalian cells. This process requires DNA-dependent protein kinase (DNA-PK), composed of heterodimeric Ku antigen and a 460,000 Da catalytic subunit (DNA-PKcs). In this study, a rabbit spinal cord model of reversible ischemia was used to demonstrate the effect of acute CNS injury on the activity and expression of DNA-dependent protein kinase. The DNA-binding activity of Ku antigen, analyzed by an electrophoretic mobility shift assay, increased during reperfusion after a short ischemic insult (15 min of occlusion), from which the animals recover neurological function. After severe ischemic injury (60 min of occlusion) and reperfusion that results in permanent paraplegia, Ku DNA binding was reduced. Protein levels of the DNA-PK componentsKu70, Ku80, and DNA-PKcswere monitored by immunoblotting. After 60 min of occlusion, the amount of DNA-PKcs and the enzyme poly(ADP-ribose) polymerase (PARP) decreased with the same time course during reperfusion. Concurrently 150 and 120 kDa fragments were immunostained by an anti-DNA-PKcs monoclonal antibody. This antibody was shown to cross-react with -fodrin breakdown products. The 120 kDa fodrin peptide is associated with caspase-3 activation during apoptosis. Both DNA-PKcs and PARP are also substrates for caspase-3-like activities. The results are consistent with a model in which after a short ischemic insult, DNA repair proteins such as DNA-PK are activated. After severe ischemic injury, DNA damage overwhelms repair capabilities, and cell death programs are initiated.

Key words: DNA damage; DNA repair; DNA-dependent protein kinase; Ku antigen; stroke; ischemia; reperfusion; spinal cord; fodrin

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Copyright © 1999 Society for Neuroscience  0270-6474/99/19124727-12$05.00/0

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