Human normal peripheral blood B-lymphocytes are deficient in DNA-dependent protein kinase activity due to the expression of a variant form of the Ku86 protein.
The heterodimeric Ku protein, which comprises a 86 kDa (Ku86) amd a 70 kDa (Ku70) subunits, is an abundant nuclear DNA-binding protein which binds in vitro to DNA termini without sequence specificity. Ku is the DNA-targeting component of the large catalytic sub-unit of the DNA-dependent protein kinase complex (DNA-PK[CS]), that plays a critical role in mammalian double-strand break repair and lymphoid V(D)J recombination. By using electrophoretic mobility shift assays, we demonstrated that in addition to the major Ku x DNA complex usually detected in cell line extracts, a second complex with faster electrophoretic mobility was observed in normal peripheral blood lymphocytes (PBL) extracts. The presence of this faster migrating complex was restricted to B cells among the circulating lymphocyte population. Western blot analysis revealed that B cells express a variant form of the Ku86 protein with an apparent molecular weight of 69 kDa, and not the 86 kDa- full-length protein. Although the heterodimer Ku70/variant-Ku86 binds to DNA-ends, this altered form of the Ku heterodimer has a decreased ability to recruit the catalytic component of the complex, DNA-PK(CS), which contributes to an absence of detectable DNA-PK activity in B cells. These data provide a molecular basis for the increased sensitivity of B cells to ionizing radiation and identify a new mechanism of regulation of DNA-PK activity that operates in vivo.
Muller
C
C
Institut de Pharmacologie et de Biologie Structurale (CNRS, UPR 9062), Toulouse, France.
Dusseau
C
C
Calsou
P
P
Salles
B
B
D034961:Antigens, Nuclear; D016175:B-Lymphocyte Subsets / Q000201:enzymology* / Q000378:metabolism; D002461:Cell Line, Transformed; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency*; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen