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Autophosphorylation of DNA-Dependent Protein Kinase Regulates DNA End Processing and May Also Alter Double-Strand Break Repair Pathway Choice

College of Veterinary Medicine and Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan 48824,¹ Departments of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1²

Received 1 August 2005/ Returned for modification 23 August 2005/ Accepted 22 September 2005

Two highly conserved double-strand break (DSB) repair pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ), function in all eukaryotes. How a cell chooses which pathway to utilize is an area of active research and debate. During NHEJ, the DNA-dependent protein kinase (DNA-PK) functions as a "gatekeeper" regulating DNA end access. Here, we provide evidence that DNA-PK regulates DNA end access via its own autophosphorylation. We demonstrated previously that autophosphorylation within a major cluster of sites likely mediates a conformational change that is critical for DNA end processing. Furthermore, blocking autophosphorylation at these sites inhibits a cell's ability to utilize the other major double-strand break repair pathway, HR. Here, we define a second major cluster of DNA-PK catalytic subunit autophosphorylation sites. Whereas blocking phosphorylation at the first cluster inhibits both end processing and HR, blocking phosphorylation at the second cluster enhances both. We conclude that separate DNA-PK autophosphorylation events may function reciprocally by not only regulating DNA end processing but also affecting DSB repair pathway choice.

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