title: Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination.
abstract: 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.
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Baeck-Seung
authors.initials: BS
authors.lastname: Lee
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Gapud
authors.forename: Shichuan
authors.initials: S
authors.lastname: Zhang
authors.forename: Yair
authors.initials: Y
authors.lastname: Dorsett
authors.forename: Andrea
authors.initials: A
authors.lastname: Bredemeyer
authors.forename: Rosmy
authors.initials: R
authors.lastname: George
authors.forename: Elsa
authors.initials: E
authors.lastname: Callen
authors.forename: Jeremy A
authors.initials: JA
authors.lastname: Daniel
authors.forename: Oleg
authors.initials: O
authors.lastname: Osipovich
authors.forename: Eugene M
authors.initials: EM
authors.lastname: Oltz
authors.forename: Craig H
authors.initials: CH
authors.lastname: Bassing
authors.forename: Andre
authors.initials: A
authors.lastname: Nussenzweig
authors.forename: Susan
authors.initials: S
authors.lastname: Lees-Miller
authors.forename: Michal
authors.initials: M
authors.lastname: Hammel
authors.forename: Benjamin P C
authors.initials: BP
authors.lastname: Chen
authors.forename: Barry P
authors.initials: BP
authors.lastname: Sleckman
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D051747:DNA-Activated Protein Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D054448:Precursor Cells, B-Lymphoid / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D060152:V(D)J Recombination
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C499308:Rag2 protein, mouse; C064658:RAG-1 protein; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D051747:DNA-Activated Protein Kinase; C497264:Prkdc protein, mouse; D004720:Endonucleases; C473337:Dclre1c protein, mouse