Lee, Baeck-Seung4; Gapud, Eric J4; Zhang, Shichuan4; Dorsett, Yair4; Bredemeyer, Andrea4; George, Rosmy4; Callen, Elsa4; Daniel, Jeremy A7; Osipovich, Oleg4; Oltz, Eugene M4; Bassing, Craig H4; Nussenzweig, Andre5; Lees-Miller, Susan4; Hammel, Michal4; Chen, Benjamin P C4; Sleckman, Barry P6
1 Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Københavns Universitet2 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Københavns Universitet3 Daniel Group, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Københavns Universitet4 unknown5 Laboratory of Genome Integrity, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA6 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA7 Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Københavns Universitet
V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA-PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA-PKcs. Mutation of these threonine residues to alanine (DNA-PKcs(3A)) renders DNA-PKcs dependent on its intrinsic kinase activity during coding end joining, at a step downstream of opening hairpin-sealed coding ends. Thus, DNA-PKcs has critical functions in coding end joining beyond promoting Artemis endonuclease activity, and these functions can be regulated redundantly by the kinase activity of either ATM or DNA-PKcs.
Molecular and Cellular Biology, 2013, Vol 33, Issue 18, p. 3568-79