Physical ARTEMIS:DNA-PKcs interaction is necessary for V(D)J recombination.

Abstract

The nuclease ARTEMIS and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are involved in the repair of physiological and pathogenic DNA double strand breaks. Both proteins are indispensable for the hairpin-opening activity in V(D)J recombination and therefore essential for the adaptive immune response. ARTEMIS and DNA-PKcs interact, however experimental evidence for in vivo significance is missing. We demonstrate that mutations abolishing this protein-protein interaction affect nuclease function. In DNA-PKcs, mutation L3062R impairs the physical interaction with ARTEMIS and was previously identified as pathogenic variant, resulting in radiosensitive severe combined immunodeficiency. In ARTEMIS, specific mutations in two conserved regions affect interaction with DNA-PKcs. In combination they impair V(D)J recombination activity, independent of ARTEMIS autoinhibitory self-interaction between the ARTEMIS C-terminus and the N-terminal nuclease domain. We describe small fragments from both proteins, capable of interaction with the corresponding full-length partner proteins: In DNA-PKcs 42 amino acids out of FAT region 2 (PKcs3041-3082) can mediate interaction with ARTEMIS. In the nuclease we have defined 26 amino acids (ARM378-403) as minimal DNA-PKcs interacting fragment. The exact mapping of the ARTEMIS:DNA-PKcs interaction may pave the way for the design of specific inhibitors targeting the repair of DNA double strand breaks.