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

Abstract

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.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

We thank Martin Gellert for many years of scientific encouragement, for perceptive suggestions, and for his meticulous review of the manuscript. Similarly, we thank Dale Ramsden for insightful discussions, sharing of ideas, and critical readings of the manuscript. We thank Nick Morrice (University of Dundee) for valiant attempts to identify additional phosphorylation sites in DNA-PKcs.

This work was supported by Public Health Service grant AI048758 (K.M.). Work in S.P.L.-M.'s laboratory is supported by grant 13639 from the Canadian Institutes of Health Research. S.P.L.-M. is supported by the Alberta Heritage Foundation for Medical Research and the Canadian Institutes of Health Research.