Gamma-irradiation directly affects the formation of coding joints in SCID cell lines

SCID mice have a defect in the catalytic subunit of the DNA-dependent prote in kinase, causing increased sensitivity to ionizing radiation in all tissu es and severely limiting the development of B and T cell lineages, SCID T a nd B cell precursors are unable to undergo normal V(D)J recombination: codi ng joint and signal joint products are less frequently formed and often wil l exhibit abnormal structural features. Paradoxically, irradiation of newbo rn SCID mice effects a limited rescue of T cell development, It is not know n whether irradiation has a direct impact on the process of V(D)J joining, or whether irradiation of the thymus allows the outgrowth of rare recombina nts. To investigate this issue, we sought to demonstrate an irradiation eff ect ex vivo. Here we have been able to reproducibly detect low-frequency co ding joint products with V(D)J recombination reporter plasmids introduced i nto SCID cell lines. Exposure of B and T lineage cells to 100 cGy of gamma irradiation made no significant difference with respect to the number of co ding joint and signal joint recombination products. However, in the absence of irradiation, the coding joints produced in SCID cells had high levels o f P nucleotide insertion. With irradiation, markedly fewer P insertions wer e seen. The effect on coding joint structure is evident in a transient assa y, in cultured cells, establishing that irradiation has an immediate impact on the process of V(D)J recombination. ri specific proposal for how the DN A-dependent protein kinase catalytic subunit influences the opening of hair pin DNA intermediates during coding joint formation in V(D)J recombination is presented.