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Murine preB lymphocytes grow in tissue culture in the presence of stromal cells and interleukin 7 (IL-7), and can be induced to differentiate to surface-immunoglobulin-positive B cells in vitro by withdrawal of IL-7. Upon differentiation, proliferation ceases, and upregulation of Rag-1 and Rag-2 expression, and induction of V(D)J immunoglobulin-gene rearrangements occur. DNA-dependent protein kinase (DNA-PK) is required for effective V(D)J recombination and repair of DNA double-strand breaks. The holoenzyme comprises a catalytic subunit (DNA-PKcs) and the Ku heterodimer (Ku70/Ku80). We have analyzed expression of Ku70, Ku80 and DNA-PKcs upon induction of differentiation in preB cells derived from wild-type, severe combined immunodeficiency (SCID) and Rag-2-/- mice. Protein levels of Ku80 and Ku70 moderately decrease after induction in all three cell types. A distinct polypeptide that crossreacts with anti-Ku Ig appears in the cytoplasm of wild-type and Rag-2-/- cells, but not of SCID cells. In mouse preB cells, Ku70 and Ku80 are present in the nuclei and cytoplasm before and after onset of differentiation. In vivo, Ku70 is predominantly expressed in V(D)J-recombination-active, early-preB and CD4-/CD8- thymocyte cell populations. Upon differentiation, protein levels of DNA-PKcs are unaltered. DNA-PK activity, which is not detectable in SCID cells, increases in wild-type and Rag-2-/- cells more than twofold shortly after induction of differentiation, then falls back to about 50% of starting levels.

Recombination protein complex RC-1, purified from calf thymus nuclear extracts, catalyzes cell-free DNA strand transfer and repair of gaps and deletions through DNA recombination. DNA polymerase E, DNA ligase III and a DNA structure-specific endonuclease co-purify with the five polypeptide complex. Here we describe the identification of two hitherto unknown subunits of RC-1. N-terminal amino acid sequences of the 160 and 130 kDa polypeptides display up to 100% identity to proteins of the structural maintenance of chromosomes (SMC) subfamilies 1 and 2. SMC proteins are involved in mitotic chromosome segregation and condensation, as well as in certain DNA repair pathways in fission (rad18 gene) and budding (RHC18 gene) yeast. The assignment was substantiated by immuno-cross-reactivity of the RC-1 subunits with polyclonal antibodies specific for Xenopus laevis SMC proteins. These antibodies, and polyclonal antibodies directed against the bovine 160 and 130 kDa polypeptides, named BSMC1 and BSMC2 (bovine SMC), inhibited RC-1-mediated DNA transfer, indicating that the SMC proteins are necessary components of the reaction. Two independent assays revealed DNA reannealing activity of RC-1, which resides in its BSMC subunits, thereby demonstrating a novel function of these proteins. To our knowledge, this is the first evidence for the association of mammalian SMC proteins with a multiprotein complex harboring, among others, DNA recombination, DNA ligase and DNA polymerase activities.

The SCID (Severe Combined Immune Deficiency) mutation causes two DNA recombination deficiencies: an aberrant joining of V(D)J immunoglobulin gene elements and a failure to perform efficient repair of DNA double-strand breaks. A recently established cell-free assay for DNA transfer (DTA) was applied to study nuclear extracts from normal and SCID-derived cells. The recombination deficiency was reflected in the cell-free system: SCID lymphocyte and fibroblast extracts showed reduced levels of DTA activity on a variety of DNA substrates. Analysis of nuclear extracts prepared from wild-type thymocytes and B cells representing different stages in lymphocyte ontogeny revealed the highest activities at the most immature stages. With progression of development, DTA activity decreased. Corresponding to their early developmental arrest, V(D)J rearrangement-incompetent RAG-2-/- lymphocyte extracts show high DTA activity. In contrast, extracts from SCID early lymphocytes express very low DNA transfer activity. Induction of V(D)J rearrangement in vivo in a normal preB cell line lead to a co-induction of the cell-free recombination activity. This indicates a development stage specificity of cell-free DNA recombination, which temporally parallels V(D)J recombination. A protein could be purified to near-homogeneity from wild-type thymocytes which stimulates the recombination activity specifically in SCID thymocyte and proB cell extracts. This protein, SRSP (SCID Recombination Stimulatory Protein), migrates as a single band of approximately 72 kDa in SDS-polyacrylamide gel electrophoresis.