Inhibition of Homologous Recombination by DNA-Dependent Protein Kinase Requires Kinase Activity, Is Titratable, and Is Modulated by Autophosphorylation

Abstract

How a cell chooses between nonhomologous end joining (NHEJ) and homologous recombination (HR) to repair a double-strand break (DSB) is a central and largely unanswered question. Although there is evidence of competition between HR and NHEJ, because of the DNA-dependent protein kinase (DNA-PK)'s cellular abundance, it seems that there must be more to the repair pathway choice than direct competition. Both a mutational approach and chemical inhibition were utilized to address how DNA-PK affects HR. We find that DNA-PK's ability to repress HR is both titratable and entirely dependent on its enzymatic activity. Still, although requisite, robust enzymatic activity is not sufficient to inhibit HR. Emerging data (including the data presented here) document the functional complexities of DNA-PK's extensive phosphorylations that likely occur on more than 40 sites. Even more, we show here that certain phosphorylations of the DNA-PK large catalytic subunit (DNA-PKcs) clearly promote HR while inhibiting NHEJ, and we conclude that the phosphorylation status of DNA-PK impacts how a cell chooses to repair a DSB.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01298-10.

ACKNOWLEDGMENTS

We thank to Ruiqiong Ye and Yao Xu for excellent technical assistance, Yaping Yu (Lees-Miller laboratory) and Rulin Zhang (WEMB Inc., Toronto, Canada) for mass spectrometry analysis of in vitro DNA-PKcs autophosphorylation sites, Jane Leitch and Diagnostics Scotland for making the phospho-specific antibody to serine 3205, and S. Elledge and P. Jeggo for sharing unpublished observations and for helpful discussions.

This work was supported by Public Health Service grant AI048758 (to K.M.) and Canadian Institutes of Health Research grant 13639 (to S.P.L.-M.).