Unique and redundant functions of ATM and DNA-PKcs during V(D)J recombination

Lymphocyte antigen receptor genes are assembled through the process of V(D)J recombination, during which pairwise DNA cleavage of gene segments results in the formation of four DNA ends that are resolved into a coding joint and a signal joint. The joining of these DNA ends occurs in G(1)-phase lymphocytes and is mediated by the non-homologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair. The ataxia telangiectasia mutated (ATM) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), two related kinases, both function in the repair of DNA breaks generated during antigen receptor gene assembly. Although these proteins have unique functions during coding joint formation, their activities in signal joint formation, if any, have been less clear. However, two recent studies demonstrated that ATM and DNA-PKcs have overlapping activities important for signal joint formation. Here, we discuss the unique and shared activities of the ATM and DNA-PKcs kinases during V(D)J recombination, a process that is essential for lymphocyte development and the diversification of antigen receptors

Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair

The nonhomologous end-joining (NHEJ) pathway is essential for radioresistance and lymphocyte-specific V(D)J (variable [diversity] joining) recombination. Defects in NHEJ also impair hematopoietic stem cell (HSC) activity with age but do not affect the initial establishment of HSC reserves. In this paper, we report that, in contrast to deoxyribonucleic acid (DNA)–dependent protein kinase catalytic subunit (DNA-PKcs)–null mice, knockin mice with the DNA-PKcs(3A/3A) allele, which codes for three alanine substitutions at the mouse Thr2605 phosphorylation cluster, die prematurely because of congenital bone marrow failure. Impaired proliferation of DNA-PKcs(3A/3A) HSCs is caused by excessive DNA damage and p53-dependent apoptosis. In addition, increased apoptosis in the intestinal crypt and epidermal hyperpigmentation indicate the presence of elevated genotoxic stress and p53 activation. Analysis of embryonic fibroblasts further reveals that DNA-PKcs(3A/3A) cells are hypersensitive to DNA cross-linking agents and are defective in both homologous recombination and the Fanconi anemia DNA damage response pathways. We conclude that phosphorylation of DNA-PKcs is essential for the normal activation of multiple DNA repair pathways, which in turn is critical for the maintenance of diverse populations of tissue stem cells in mice

Regulation of DNA Repair by Atm and DNA-PKcs during V9D)J Recombination

V(D)J recombination is the somatic rearrangement process that through which the genetic diversity of antigen receptors is established during lymphocyte development. DNA double strand breaks (DSBs) induced in G1-phase of the cell cycle are necessary intermediates in this process. Accurate and efficient resolution of these lesions is necessary not only for achieving a broad repertoire of antigen recognition specificities, but also for preventing genomic instability that could result in oncogenic transformation events. Atm and DNA-PKcs are ubiquitously expressed proteins that transduce and orchestrate responses to DNA double strand breaks (DSBs) of genotoxic or physiologic origin in G1-phase cells. Each protein respectively is known to have unique functions during the repair of coding ends. I demonstrate here, however, that the kinase activities of Atm and DNA-PKcs redundantly drive normal and efficient coding joint formation. Furthermore, I also show that Atm and DNA-PKcs redundantly promote efficient processing and repair of signal ends both within the chromosome and on extrachromosomal excision circles. The overlapping functions of these two proteins during signal end repair also depend on redundant kinase activities. I also investigate the end processing and joining activities of potential shared substrates of the Atm and DNA-PKcs kinases during V(D)J recombination in order to better understand how Atm and DNA-PKcs orchestrate normal repair of both coding and signal ends. These candidate substrates include RAG-1 and RAG-2, which together comprise the nuclease that initiates V(D)J recombination, the chromatin-associated DNA repair factor 53BP1, and the DNA-PKcs protein itself. My findings indicate that Atm and DNA-PKcs have significantly broader roles in iii V(D)J recombination than previously appreciated. Moreover, these results have important implications for the general understanding of non-homologous end joining (NHEJ)-mediated DNA repair

Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination

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