The inability to repair double strand breaks (DSB) in DNA underlies genetic instability, cancer predisposition, and immune dysfunction. A new DNA repair protein, hPso4, has recently been identified through its association with the V(D)J recombination protein terminal deoxynucleotidyl transferase (TdT). The ubiquitous expression of hPso4, its induction following exposure to DSB-inducing agents, and its requirement for clonogenic survival following y-irradiation strongly suggest a role for this protein in DSB repair. This proposal will investigate the specific role of hPso4 in DNA end joining and at sites of V(D)J recombination and examine the consequences of its deficiency for the immune system and for oncogenesis. First, the requirement of hPso4 for DNA repair will be examined in cell systems to define its role in end joining. Specifically, it will be determined whether hPso4 interacts with the core proteins required for non-homologous end joining reactions. The association of hPso4 with TdT will also be investigated to determine its role in modulating TdT activity during V(D)J recombination. Next, the question of whether hPso4, a protein whose yeast homolog is known to have E3 ubiquitin ligase activity, undergoes post-translational modification (phosphorylation and/or ubiquitination) and whether such modification(s) affect the ability of Pso4 to interact with or modify other DNA repair) proteins will be examined. Finally, a mouse model of Pso4 deficiency will be enerated to explore its potential biologic role in maintaining DNA integrity and promoting normal lymphoid development. Specific questions to be asked of this model are: do Pso4 -/- mice have developmental abnormalities, aberrations in B or T lymphocyte development, defects in V(D)] rearrangements, and/or an increased incidence of lymphoid or other malignancies? PSO4-/- murine cell lines will be scored for susceptibility to y-irradiation and chemical mutagens as compared with and littermate controls and murine cell lines deficient in other DNA repair proteins. The results of these studies will provide insights into the mechanisms by which Pso4 promotes DNA repair in mammalian cells and define whether it has a major biologic role in the V(D)J recombination pathway or in enhancing genomic stability. ? ?
