The Functional Role of the ARTEMIS-DNA-PKcs Interaction in the Repair of Physiological and Pathological DNA Double Strand Breaks

• Applicant: Dr. Klaus Schwarz
• Subject Area: General Genetics and Functional Genome Biology ; Immunology ; Cell Biology
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 408613301

The nuclease ARTEMIS and the protein kinase DNA-PKcs are proteins involved in the repair of both physiological and pathological DNA double strand breaks (DSB). They are part of the canonical Non-Homologous End-Joining (cNHEJ) pathway. ARTEMIS and DNA-PKcs can interact and together they constitute the hairpin-opening activity in V(D)J recombination, which is essential for the development of B and T lymphocytes of the adaptive immune system. Loss of either protein results in radio-sensitive severe combined immunodeficiency (RS-SCID), a syndrome which will lead to death in the first year of life, if left untreated. ARTEMIS mutations are a frequent cause of RS-SCID, however only very few patients are known to have a mutation in DNA-PKcs. The phenotype of the RS-SCID patient harbouring the disease causing point mutation pLeu3062Arg in DNA-PKcs resembles ARTEMIS-null patients. In vitro, full length ARTEMIS is phosphorylated by DNA-PKcs and it has DNA-PKcs dependent hairpin-opening activity. Up to now the in vivo significance of the ARTEMIS-DNA-PKcs interaction and of the DNA-PKcs-dependent phosphorylation of ARTEMIS is unclear. Mutations in the major phosphorylation sites in the ARTEMIS C-terminus do not affect ARTEMIS function, neither in V(D)J recombination nor in general DSB repair. Our preliminary results show that amino acid exchange pLeu3062Arg in DNA-PKcs impairs the interaction with ARTEMIS, suggesting this could be causative of the RS-SCID phenotype. In this project we will use a genetic and a biochemical approach to elucidate whether the interaction between ARTEMIS and DNA-PKcs is a necessary prerequisite for V(D)J recombination and the repair of IR-induced DSB. Using CRISPR/Cas 9 technology we will introduce specific point mutations in the protein-protein interaction domains of the endogenous proteins and subsequently test the effect on ARTEMIS function in V(D)J recombination and DNA repair assays. In a second approach, we will use peptides derived from the protein-protein interaction domains and test their ability to block ARTEMIS function in vivo. DNA-PKcs is a multifunctional protein involved in diverse signal transduction pathways. With the two independent experimental strategies it will be possible to conclusively evaluate in vivo the functional role of the ARTEMIS-DNA-PKcs interaction in cNHEJ. In the future the results of the biochemical approach can be used as starting point for the development of peptides or peptidomimetics for targeting the specific protein-protein interaction in ARTEMIS-dependent DSB repair in distinct malignancies.

• DFG Programme: Research Grants