A DNA-PKcs mutation in a radiosensitive T–B– SCID patient inhibits Artemis activation and nonhomologous end-joining
Mirjam van der Burg, Hanna IJspeert, Nicole S. Verkaik, Tuba Turul, Wouter W. Wiegant, Keiko Morotomi-Yano, Pierre-Olivier Mari, Ilhan Tezcan, David J. Chen, Malgorzata Z. Zdzienicka, Jacques J.M. van Dongen, Dik C. van Gent
Mirjam van der Burg, Hanna IJspeert, Nicole S. Verkaik, Tuba Turul, Wouter W. Wiegant, Keiko Morotomi-Yano, Pierre-Olivier Mari, Ilhan Tezcan, David J. Chen, Malgorzata Z. Zdzienicka, Jacques J.M. van Dongen, Dik C. van Gent
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Research Article Immunology

A DNA-PKcs mutation in a radiosensitive T–B– SCID patient inhibits Artemis activation and nonhomologous end-joining

Abstract

Radiosensitive T–B– severe combined immunodeficiency (RS-SCID) is caused by defects in the nonhomologous end-joining (NHEJ) DNA repair pathway, which results in failure of functional V(D)J recombination. Here we have identified the first human RS-SCID patient to our knowledge with a DNA-PKcs missense mutation (L3062R). The causative mutation did not affect the kinase activity or DNA end-binding capacity of DNA-PKcs itself; rather, the presence of long P-nucleotide stretches in the immunoglobulin coding joints indicated that it caused insufficient Artemis activation, something that is dependent on Artemis interaction with autophosphorylated DNA-PKcs. Moreover, overall end-joining activity was hampered, suggesting that Artemis-independent DNA-PKcs functions were also inhibited. This study demonstrates that the presence of DNA-PKcs kinase activity is not sufficient to rule out a defect in this gene during diagnosis and treatment of RS-SCID patients. Further, the data suggest that residual DNA-PKcs activity is indispensable in humans.

Authors

Mirjam van der Burg, Hanna IJspeert, Nicole S. Verkaik, Tuba Turul, Wouter W. Wiegant, Keiko Morotomi-Yano, Pierre-Olivier Mari, Ilhan Tezcan, David J. Chen, Malgorzata Z. Zdzienicka, Jacques J.M. van Dongen, Dik C. van Gent

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Figure 6

Residual function of DNA-PKcs defect.

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Residual function of DNA-PKcs defect. (A) Immunostaining of microirradia...
(A) Immunostaining of microirradiated wild-type and delG2113/L3062R YFP-DNA-PKcs–expressing V3 cells after cotransfection with a wild-type myc-Artemis expression construct (in red). Cells were microirradiated using a multiphoton laser system. One of the two V3 cells expressing delG2113/L3062R YFP-DNA-PKcs was not transfected with Artemis. This figure shows representative results of 2 independent experiments, each with multiple laser-damaged cells. (B) DNA end-joining assay in DNA-PKcs–deficient V3 cells and V3 cells expressing wild-type or ID177 mutant DNA-PKcs. Linearized pDVG94 can be rejoined via direct or microhomology-directed end-joining. Joining via microhomology results in the generation of a BstXI restriction site (36). Junctions were PCR amplified, and PCR products were digested with BstXI, as indicated.