R. Fukumura et al., Signal joint formation is also impaired in DNA-dependent protein kinase catalytic subunit knockout cells, J IMMUNOL, 165(7), 2000, pp. 3883-3889
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.