Signal joint formation is also impaired in DNA-dependent protein kinase catalytic subunit knockout cells

The effort to elucidate the mechanism of V(D)J recombination has given rise to a dispute as to whether DNA-dependent protein kinase catalytic subunit (DNA-PKcs) contributes to signal joint formation (sjf). Observations report ed to date are confusing. Analyses using DNA-PKcs-deficient cells could not conclude the requirement of DNA-PKcs for sjf, because sjf can be formed by end-joining activities which are diverse among cells other than those part icipating in V(D)J recombination. Here, we observed V(D)J recombination in DNA-PKcs knockout cells and showed that both signal and coding joint format ion were clearly impaired in the cells. Subsequently, to directly demonstra te the requirement of DWA-PKcs for sjf, we introduced full-length cDNA of D NA-PKcs into the knockout cells. Furthermore, several mutant DNA-PKcs cDNA constructs designed from mutant cell lines (irs20, V3, murine scid, and SX9 ) were also introduced into the cells to obtain further evidence indicating the involvement of DNA-PKcs in sjf, We found as a result that the full-len gth cDNA complemented the aberrant sjf and that the mutant cDNAs constructs also partially complemented it. Lastly, we looked at whether the kinase ac tivity of DNA-PKcs is necessary for sjf and, as a result, demonstrated a cl ose relationship between them. Our observations clearly indicate that the D NA-PKcs controls not only coding joint formation but also the sjf in V(D)J recombination through its kinase activity.