V(D)J site-specific recombination is the process that establishes diversity of the immune repertoire by mediating the somatic assembly of the antigen receptor gene segments. Mutations within the components that catalyze the V(D)J reaction result in severe immunodeficiencies. Two of these components, the recombination activating proteins, Rag-1 and Rag-2, are the key lymphoid specific activities that initiate the catalysis of the process. In addition, several ubiquitous, DNA repair activities have been identified that also participate in the V(D)J process. The long-term objectives of this application are to elucidate the biochemistry of V(D)J recombination.
The specific aims are:
Aim 1 will address the structural domains and the biochemical properties of Rag-1 and Rag-2.
Aim 2 will analyze in detail the assembly and reaction kinetics of the individual components that are required in order to form a V(D)J recombination competent complex. Studies in aims 1 and 2 will be based on conventional biochemical techniques and modern technology using a biosensor instrument that can monitor interactions between macromolecules in real time. Purified Rag-1, Rag-2 and ubiquitous DNA repair activities will be assayed for their DNA binding and protein/protein interaction activities using footprinting, cross-linking and biosensor assays. In addition, the role of the identified domains will be tested in vivo using genetic approaches. The DNA binding domain of Rag-1 will be crystallized in order to understand its mode of binding.
Aim 3 will address the catalytic domain of Rag-1/Rag-2 and how the Rag complex cleaves the DNA with specificity. These experiments will employ genetic analyses, chemical modifications and mass spectroscopy techniques using purified Rag-1 and Rag-2 proteins. Studies on the biochemistry and regulation of V(D)J recombination are of profound significance for our understanding of how the immune system is established and how immunodeficiencies in humans can be generated by the stochastic inactivation of the V(D)J recombination machinery. In addition, V(D)J recombination has been implicated in oncogenesis and the translocation of certain oncogenes. The studies described in this proposal may help us design reagents and experimental protocols to diagnose many debilitating or fatal diseases of the human lymphopoietic system such as, forms of severely combined immunodeficiencies, leukemias and lymphomas. A subset of these diseases may be treatable by gene therapy.
|Gomez, C A; Ptaszek, L M; Villa, A et al. (2000) Mutations in conserved regions of the predicted RAG2 kelch repeats block initiation of V(D)J recombination and result in primary immunodeficiencies. Mol Cell Biol 20:5653-64|
|Aidinis, V; Dias, D C; Gomez, C A et al. (2000) Definition of minimal domains of interaction within the recombination-activating genes 1 and 2 recombinase complex. J Immunol 164:5826-32|
|Aidinis, V; Bonaldi, T; Beltrame, M et al. (1999) The RAG1 homeodomain recruits HMG1 and HMG2 to facilitate recombination signal sequence binding and to enhance the intrinsic DNA-bending activity of RAG1-RAG2. Mol Cell Biol 19:6532-42|
|Santagata, S; Besmer, E; Villa, A et al. (1999) The RAG1/RAG2 complex constitutes a 3' flap endonuclease: implications for junctional diversity in V(D)J and transpositional recombination. Mol Cell 4:935-47|
|Santagata, S; Aidinis, V; Spanopoulou, E (1998) The effect of Me2+ cofactors at the initial stages of V(D)J recombination. J Biol Chem 273:16325-31|
|Villa, A; Santagata, S; Bozzi, F et al. (1998) Partial V(D)J recombination activity leads to Omenn syndrome. Cell 93:885-96|