Analyses of TCRB rearrangements substantiate a profound deficit in recombination signal sequence joining in SCID foals: Implications for the role of DNA-dependent protein kinase in V(D)J recombination

We reported previously that the genetic SCID disease observed in Arabian fo als is explained by a defect in V(D)J recombination that profoundly affects both coding and signal end joining. As in C,B-17 SCID mice, the molecular defect in SCID foals is in the catalytic subunit of the DNA-dependent prote in kinase (DNA-PKCS); however, in SCID mice, signal end resolution remains relatively intact. Moreover, recent reports indicate that mice that complet ely lack DNA-PKCS also generate signal joints at levels that are indistingu ishable from those observed in C,B-17 SCID mice, eliminating the possibilit y that a partially active version of DNA-PKCS facilitates signal end resolu tion in SCID mice. We have analyzed TCRB rearrangements and find that signa l joints are reduced by similar to 4 logs in equine SCID thymocytes as comp ared with normal horse thymocytes. A potential explanation for the differen ces between SCID mice and foals is that the mutant DNA-PKCS allele in SCID foals inhibits signal end resolution. We tested this hypothesis using DNA-P RCS expression vectors; in sum, we find no evidence of a dominant-negative effect by the mutant protein, These and other recent data are consistent wi th an emerging consensus: that in normal cells, DNA-PKCS participates in bo th coding and signal end resolution, but in the absence of DNA-PKCS an unde fined end joining pathway (which is variably expressed in different species and cell types) can facilitate imperfect signal and coding end joining.