A DNA-PKcs mutation in a radiosensitive T–B– SCID patient inhibits Artemis activation and nonhomologous end-joining
Mirjam van der Burg, … , Jacques J.M. van Dongen, Dik C. van Gent
Mirjam van der Burg, … , Jacques J.M. van Dongen, Dik C. van Gent
Published December 15, 2008
Citation Information: J Clin Invest. 2009;119(1):91-98. https://doi.org/10.1172/JCI37141.
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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 5

Confirmation of disease-causing effect of DNA-PKcs mutation.

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Confirmation of disease-causing effect of DNA-PKcs mutation. (A) Clonoge...
(A) Clonogenic survival assay of DNA-PKcs–deficient V3 cells (57) complemented with wild-type DNA-PKcs or mutated DNA-PKcs (delG2113/L3062R) compared with wild-type (AA8) cells. In contrast to wild-type DNA-PKcs, mutated DNA-PKcs was not able to complement the radiosensitivity of V3 cells. Error bars represent the SD from 3 independent experiments. (B) Sequence alignment of DNA-PKcs protein sequences of different species. G2113 is located in a region without a predicted secondary structure (indicated as –); L3062 is located in a predicted helix (H). PredictProtein analysis software was used. (C) Coding end structure of recombination substrate (pDVG93) used in the V(D)J recombination assay (31). Depending on the manner of hairpin opening and coding joint formation, 3 different types of coding joints can be generated and discriminated by restriction enzyme digestion. Coding joints with long stretches of homologous P-nucleotides without nucleotide loss results in an NgoMI restriction site. A 4 bp microhomology of the coding ends creates a NotI restriction site. Junctions that have been processed differently will not contain either of the 2 restriction sites. (D) PCR and restriction enzyme digestion of coding joints recovered from DNA-PKcs–deficient V3 cells and V3 cells expressing wild-type DNA-PKcs or mutated DNA-PKcs after cotransfection of pDVG93 and RAG1 and RAG2 expression constructs. Wild-type DNA-PKcs and DNA-PKcs delG2113 were able to restore the shifted junction pattern as observed in DNA-PKcs–deficient V3 cells.