During development of the immune system, antigen receptor genes in B and T cells are assembled from germline coding gene segments by a site-specific somatic recombination process termed V(D)J recombination. Defects in this pathway result in a number of primary immunodeficiencies such as Severe Combined Immuno Deficiency (SCID) and Omenn Syndrome and have the potential to result in development of leukemias and lymphomas. To prevent development of such diseases, there is a need to properly resolve the DNA intermediates generated during the recombination reaction. These include DNA double strand breaks (DSB) that must be repaired by the NHEJ pathway, and DNA hairpin structures that must be opened and processed during V(D)J recombination. Mutations in a novel protein called Artemis causes RS-SCID syndrome, a SCID phenotype associated with radiosensitivity. Data from in vitro analysis and knockout mice have shown that Artemis is the nuclease that opens hairpin-coding ends during V(D)J recombination. However, little is known about the biochemistry and structure of Artemis. Further, direct evidence for its role in NHEJ is still lacking. Furthermore, the C-terminal region of Artemis, which constitutes almost half of the protein, remains poorly characterized and the contribution of Artemis- endonucleasedefective mutants to V(D)J recombination has also not been analyzed. In this grant application, we describe a series of experiments that will address these issues while extending observations we have regarding multiple and yet uncharacterized roles of Artemis in V(D)J recombination and DNA repair. Through specific aims proposed we will characterize the biochemical properties of Artemis (oligomerization, DNA binding and nuclease activity) and develop its crystal structure (aim 1), investigate the in vivo relevance of novel protein interactions and multiple functions of Artemis in NHEJ (aim2) and V(D)J recombination (aim 3). Together, the experiments proposed in the specific aims will begin to define the structure-function relationship of Artemis and the mechanism of different RS-SCID mutations found in humans. Further, by establishing the importance of novel protein interactions of Artemis and its multiple functions in both V(D)J recombination and NHEJ, we will provide new insights into the molecular mechanism of these processes. Through this study, we will also describe the protein structure of Artemis and further define its role(s) in development of a healthy immune system.
This project focuses on the study of Artemis, a protein that is essential for the generation of the immune system. Absence of Artemis leads to RS-SCID, a Severe Combined Immuno-Deficiency associated with radiosensitivity and Omenn Syndrome. Artemis was cloned in 2001 and despite its central role in V(D)J recombination and genomic stability, the multiple roles that this factors has, its structure and structuralfunctional relationship remains largely unexplored. Our preliminary studies show that we can produce large quantities of highly pure and active recombinant Artemis protein as well as its C-terminal domain. Proposed experiments will investigate the biochemical and structural properties of these pure recombinant proteins. Efforts are also described to express, purify and analyze the N-terminal domain of Artemis. Additional preliminary data describe a novel function for Artemis C-terminal region, and suggest the generation of an anomalous repertoire of immune cells in patients carrying Artemis? mutations. These findings will be further explored to obtain a clear understanding of the multiple roles of Artemis in V(D)J recombination, DNA repair and genomic stability. In summary, by increasing our knowledge of Artemis? function these studies will contribute to our understanding of human diseases, specifically, immunodeficiencies and cancer and will facilitate the path to find better treatments for these life-threatening diseases.
| De Ioannes, Pablo; Malu, Shruti; Cortes, Patricia et al. (2012) Structural basis of DNA ligase IV-Artemis interaction in nonhomologous end-joining. Cell Rep 2:1505-12 |
| Malu, Shruti; De Ioannes, Pablo; Kozlov, Mikhail et al. (2012) Artemis C-terminal region facilitates V(D)J recombination through its interactions with DNA Ligase IV and DNA-PKcs. J Exp Med 209:955-63 |
| Malu, Shruti; Malshetty, Vidyasagar; Francis, Dailia et al. (2012) Role of non-homologous end joining in V(D)J recombination. Immunol Res 54:233-46 |
