STIMULATION OF DEFECTIVE-DNA TRANSFER ACTIVITY IN RECOMBINATION DEFICIENT SCID CELL-EXTRACTS BY A 72-KDA PROTEIN FROM WILD-TYPE THYMOCYTES

The SCID (Severe Combined Immune Deficiency) mutation causes two DNA r ecombination 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 transfe r (DTA) was applied to study nuclear extracts from normal and SCID-der ived cells. The recombination deficiency was reflected in the cell-fre e system: SCID lymphocyte and fibroblast extracts showed reduced level s of DTA activity on a variety of DNA substrates. Analysis of nuclear extracts prepared from wild-type thymocytes and B cells representing d ifferent stages in lymphocyte ontogeny revealed the highest activities at the most immature stages. With progression of development, DTA act ivity 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 expre ss 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-fr ee recombination activity. This indicates a development stage specific ity of cell-free DNA recombination, which temporally parallels V(D)J r ecombination. A protein could be purified to near-homogeneity from wil d-type thymocytes which stimulates the recombination activity specific ally in SCID thymocyte and proB cell extracts. This protein, SRSP (SCI D Recombination Stimulatory Protein), migrates as a single band of app roximately 72 kDa in SDS-polyacrylamide gel electrophoresis.