The C-terminal conserved domain of DNA-PKcs, missing in the SCID mouse, is required for kinase activity.

DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact. The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity. Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity. We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells. Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase. We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines.

Targeted disruption of the catalytic subunit of the DNA-PK gene in mice confers severe combined immunodeficiency and radiosensitivity.

The DNA-dependent protein kinase is a mammalian protein complex composed of Ku70, Ku80, and DNA-PKcs subunits that has been implicated in DNA double-strand break repair and V(D)J recombination. Here, by gene targeting, we have constructed a mouse with a disruption in the kinase domain of DNA-PKcs, generating an animal model completely devoid of DNA-PK activity. Our results demonstrate that DNA-PK activity is required for coding but not for signal join formation in mice. Although our DNA-PKcs defective mice closely resemble Scid mice, they differ by having elevated numbers of CD4+CD8+ thymocytes. This suggests that the Scid mice may not represent a null phenotype and may retain some residual DNA-PKcs function.

Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20.

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases. A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain. Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination. Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than two previously characterised mutants, V-3 and mouse scid cells. Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but is unable to function as a protein kinase. To verify the defect in irs-20 cells and provide insight into the function and expression of DNA-PKcs in double-strand break repair and V(D)J recombination we introduced YACs encoding human and mouse DNA-PKcs into defective mutants and achieved complementation of the defective phenotypes. Furthermore, in irs-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus. This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is important for protein kinase activity.

Accumulation of HL-60 leukemia cells in G2/M and inhibition of cytokinesis caused by two marine compounds, bistratene A and cycloxazoline.

The effects on the cell cycle of two biologically active compounds, bistratene A and cycloxazoline, from the marine ascidian Lissoclinum bistratum were studied in HL-60 human leukemia cells using flow cytometry. Both compounds were shown to cause an apparent accumulation of cells in the G2/M phase. This effect was shown to be both time- and dose-dependent. At the longer time points (30 and 48 h after addition of the compounds) polyploidy was apparent. The fate of cells labeled in the S phase with 5-bromo-2'-deoxyuridine (BrdUrd) was analysed using a bivariate BrdUrd/PI (propidium iodide) technique. Bistratene A and cycloxazoline treatment prevented the majority of BrdUrd-labeled cells from progressing through to the G1 phase. Approximately 50% of the cells were delayed at G2/M, and a significant proportion of cells appeared to be polyploid. Light and electron microscopy revealed the presence of multinucleated cells accounting for the apparent polyploidy. The progression of cells out of the G1 phase was also examined by synchronising cells with mimosine and releasing them from mimosine block in the presence of bistratene A. There was no evidence of a block at the G1/S phase transition or through the S phase since DNA synthesis was not inhibited. The mechanism by which these compounds interfere with cytokinesis is presently unknown but, in the case of bistratene A, may be linked to altered phosphorylation of cellular proteins involved in cell-cycle control.