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 reported 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 participating 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 formation were clearly impaired in the cells. Subsequently, to directly demonstrate the requirement of DNA-PKcs for sjf, we introduced full-length cDNA of DNA-PKcs into the knockout cells. Furthermore, several mutant DNA-PKcs cDNA constructs designed from mutant cell lines (irs-20, 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-length cDNA complemented the aberrant sjf and that the mutant cDNAs constructs also partially complemented it. Lastly, we looked at whether the kinase activity of DNA-PKcs is necessary for sjf and, as a result, demonstrated a close relationship between them. Our observations clearly indicate that the DNA-PKcs controls not only coding joint formation but also the sjf in V(D)J recombination through its kinase activity.
Fukumura
R
R
National Institute of Radiological Sciences, Chiba, Japan.
Araki
R
R
Fujimori
A
A
Tsutsumi
Y
Y
Kurimasa
A
A
Li
G C
GC
Chen
D J
DJ
Tatsumi
K
K
Abe
M
M
D000818:Animals; D020134:Catalytic Domain / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics; D005786:Gene Expression Regulation; D015321:Gene Rearrangement; D016654:Genes, RAG-1; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000096:biosynthesis / Q000138:chemical synthesis; D051379:Mice; D018345:Mice, Knockout; D016513:Mice, SCID; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D011995:Recombination, Genetic; D014162:Transfection
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D021382:Protein Sorting Signals; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases