Signal joint formation is inhibited in murine scid preB cells and fibroblasts in substrates with homopolymeric coding ends

Abstract During B and T lymphocyte development, immunoglobulin and T cell receptor genes are assembled from the germline V, (D) and J gene segments (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27–150). These DNA rearrangements, responsible for immune system diversity, are mediated by a site specific recombination machinery via recognition signal sequences (RSSs) composed of conserved heptamers and nonamers separated by spacers of 12 or 23 nucleotides (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27–150). Recombination occurs only between a RSS with a 12mer spacer and a RSS with a 23mer spacer (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27–150). RAG1 and RAG2 proteins cleave precisely at the RSS-coding sequence border leading to flush signal ends and coding ends with a hairpin structure (Eastman, M., Leu, T., Schatz, D., 1996. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature 380, 85–88; Roth, D.B., Menetski, J.P., Nakajima, P.B., Bosma, M.J., Gellert, M., 1992. V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell 983–991; Roth, D.B., Zhu, C., Gellert, M., 1993. Characterization of broken DNA molecules associated with V(D)J recombination. Proc. Natl. Acad. Sci. USA 90, 10,788–10,792; van Gent, D., McBlane, J., Sadofsky, M., Hesse, J., Gellert, M., 1995. Initiation of V(D)J recombination in a cell-free system. Cell 81, 925–934). Signal ends join, forming a signal joint. The hairpin coding ends are opened by a yet unknown endonuclease, and are further processed to form the coding joint (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Ad. Immunol. 56, 27–150.) The murine scid mutation has been shown to affect coding joints, but much less signal joint formation. In this study we demonstrate that the murine scid mutation inhibits correct signal joint formation when both coding ends contain homopolymeric sequences. We suggest that this finding may be due to the function of the SCID protein as an assembly component in V(D)J recombination.