Molecular mechansims of Artemis mediated immunodeficiencies

V(D)J recombination is a site specific recombination reaction that occurs in developing B and T cells and is essential for their survival and function. ARTEMIS was discovered as a factor critical for this process through presence of its mutations in human radiosensitive severe combined immune deficiency (RS-SCID) syndrome. It is known to open hairpin coding end intermediates during V(D)J recombination, and is also a Non- Homologous End-Joining (NHEJ) factor required for DNA repair. During our search for novel interactions of Artemis with other NHEJ factors, we found that Ligase IV, another critical player of these processes interacts with Artemis in human pre B cells. This interaction was mediated by Artemis' C-terminal region and the DNA binding domain of Ligase IV. Amino acids within the C-terminal region of Artemis that are critical for this interaction were identified and shown to be required for efficient V(D)J recombination and resistance to ionizing radiations. The hypomorphic human mutations within the C-terminal region of Artemis that cause combined SCID plus development of B cell lymphomas also fail to interact with Ligase IV. Thus, for the first time we show a direct role of the C-terminal region of Artemis in V(D)J recombination and NHEJ and the mechanism by which these hypomorphic mutations of Artemis may cause immunodeficiency and cancer. Sequence analysis of Artemis has predicted a number of residues within its N-terminal region to be critical for its endonuclease function. We have found another highly conserved residue within Artemis, threonine 65, by identification of its missense mutation in a patient suffering from radiosensitive Atypical SCID/Omenn Syndrome. This mutation (T65I) results in decreased Artemis protein levels in vivo, defective endonuclease function in vitro and significant reduction in levels of V(D)J recombination on a plasmid substrate based assay. Coding joints generated in presence of this mutation and a SCID mutation of Artemis, D165N showed decreased junctional processing, providing evidence for Artemis' role as the nuclease that trims the coding ends before joining. Thus, highly reduced V(D)J recombination and defective generation of antigen receptors that may induce deletion of immature lymphocytes and/or receptor editing, may be the mechanisms explaining the etiology of this Artemis mediated immunodeficiency.