Processing of Mammalian Episomal Substrates by Hypomorphic Artemis Mutants and Role of DNA-PKcs Phosphorylation

Background: Inherited hypomorphic Artemis alleles have been identified in patients that cause combined immunodeficiency syndromes of varying severity. Characteristically, these are premature translation termination mutants (D451X, T432X, S385X; where X represents stop codon) resulting in either full or partial loss of C terminus. Functional evidence exists, suggesting a role of these hypomorphic mutants in impairing general double-strand break (DSB) repair bringing about genomic instability and protumorigenic chromosomal rearrangements; a discrete function outside of its canonical function in V(D)J recombination. Here, we characterize the effect of these mutants on episomal end-joining substrates in a model system for DSBs induced in a near physiological environment. Results: We employed replica plating assay to determine the effect on repair fidelity of endjoining upon overexpression of different Artemis variants. We found, markedly increase nuclease activity of the S385X (βCASP) mutant resulting in increase in number of episomes with truncations. Further, we sought to determine the effect of inhibition of DNA-PKcs phosphorylation (T2609 cluster) in regulation of episomal repair fidelity in cell lines expressing Artemis mutants. Upon inhibition of phosphorylation, we found out reduced number episomes with truncations resulting in increase in repair fidelity. Conclusions: Our work provides a novel venue to study the effects of Artemis mutants using an episomal system containing an inducible DSB. Our work indicates that the S385X deletion is more nucleolytically active probably because it is less regulated (lacks C terminal domain). We also provide evidence for an important role of DNA-PKcs in facilitating endonuclease activity of hypomorphic Artemis mutants.