Abstract:A 150-kDa DNA-dependent ATPase composed of 83/68-kDa subunits was previously reported to cofractionate with a 21S complex of enzymes for DNA synthesis from HeLa cells (Vishwanatha, J. K., & Baril, E. F. (1990) Biochemistry 29, 8753-8759). The DNA-dependent ATPase was purified to electrophoretic homogeneity from a HeLa cell homogenate by a modified procedure that involves subcellular fractionation, poly(ethylene-glycol) precipitation of the combined nuclear extract/cytosol, and chromatography on Q-Sepharose and… Show more
“…The tight association of these enzymatic activities with DNA are reminiscent of a DNA-dependent protein kinase/ATPase complex detected in HeLa cells (15)(16)(17)(18). The latter complex has been suggested to be involved in the regulation of transcription, DNA replication and cell growth (17).…”
Section: Discussionmentioning
confidence: 99%
“…The latter complex has been suggested to be involved in the regulation of transcription, DNA replication and cell growth (17). Although the physical constants ofthe previously described complex are clearly different (16)(17)(18), it is of interest that a combined protein kinase/ATPase activity has been correlated with DNA transcription and replication (17), functions that are also expected to be associated with the bases of DNA loops (2,3).…”
Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller,S.A., Dykes,D.D. and
“…The tight association of these enzymatic activities with DNA are reminiscent of a DNA-dependent protein kinase/ATPase complex detected in HeLa cells (15)(16)(17)(18). The latter complex has been suggested to be involved in the regulation of transcription, DNA replication and cell growth (17).…”
Section: Discussionmentioning
confidence: 99%
“…The latter complex has been suggested to be involved in the regulation of transcription, DNA replication and cell growth (17). Although the physical constants ofthe previously described complex are clearly different (16)(17)(18), it is of interest that a combined protein kinase/ATPase activity has been correlated with DNA transcription and replication (17), functions that are also expected to be associated with the bases of DNA loops (2,3).…”
Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller,S.A., Dykes,D.D. and
“…Biochemical analyses of Ku demonstrated that it bound in vitro to DNA termini or other discontinuities in the DNA structure without apparent sequence speciÂźcity (Minori and Hardin, 1986;Blier et al, 1993;Falzon et al, 1993). Ku has also been shown to possess DNA-dependent ATPase (Cao et al, 1994) and helicase (Tuteja et al, 1994) activities. Lastly, Ku has also been reported to be capable of sequence-speciÂźc binding within the promoter elements of genes (Gri n et al, 1996).…”
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 speciÂźcity. 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 doublestrand break repair and lymphoid V(D)J recombination. By using electrophoretic mobility shift assays, we demonstrated that in addition to the major Kuâ 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.
“…22 Biochemical analyses of Ku demonstrated that it bound in vitro to DNA termini in the DNA structure without apparent sequence specificity. [23][24][25] Ku has also been shown to possess DNA-dependent adenosine triphosphatase 26 and helicase 27 activities. In vitro, heterodimerization of active Ku requires the cotranslation of both subunits.…”
Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation. The purpose of this study was to evaluate the radiosensitivity and DSB repair after transfection of a Ku86-antisense in a human fibroblast cell line. Simian virus 40-transformed MRC5V1 human fibroblasts were transfected with a vector (pcDNA3) containing a Ku86-antisense cDNA. The main endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kinase activity, clonogenic survival, and DSB repair kinetics. After transfection of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-binding activity, and DNA protein kinase activity were observed in the uncloned cellular population. The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observed at 24 hours after irradiation compared with 0% for the control. Several clones were also isolated with a decreased level of Ku86 protein, a surviving fraction at 2 Gy between 0.05 and 0.40, and 10 -20% of unrepaired DSB at 24 hours. This study is the first to show the implication of Ku86 in DSB repair and in the radiosensitivity of human cells. This investigation strongly suggests that Ku86 could constitute an appealing target for combining gene therapy and radiation therapy. Cancer Gene Therapy (2000) 7, 339 -346
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