DNA-dependent protein kinase defects are linked to deficiencies in DNA repair and V(D)J recombination.

DNA-dependent protein kinase is a nuclear serine/threonine kinase whose catalytic properties are expressed only when the enzyme is bound to DNA ends or other discontinuities in the DNA. DNA-PK comprises two components: one mediates binding to DNA and corresponds to the heterodimeric human autoimmune antigen Ku; the other, DNA-PK catalytic subunit (DNA-PKcs), is a polypeptide of approximately 450 kDa. DNA-PK deficiencies are associated with certain mutant rodent cell lines that display defects in DNA double strand break repair and V(D)J recombination. Specifically, hamster xrs-6 cells lack Ku function, whereas murine scid and hamster V3 cells lack functional DNA-PKcs. Furthermore, the phenotypes of xrs-6 and V3 cells can be corrected by the expression of the genes encoding the 80 kDa component of Ku or DNA-PKcs, respectively. These results imply that DNA-PK is an important component of the DNA double strand break repair/recombination apparatus. Possible roles for DNA-PK in these processes are discussed.

DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair.

DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase composed of a catalytic subunit called p350 and a DNA binding component termed Ku. Ku consists of two tightly associated polypeptides of approximately 70 kDa and 80 kDa (Ku80). An intriguing feature of DNA-PK is that it binds to DNA ends and other discontinuities in DNA and requires these structures for its activation. This suggests that DNA-PK may function in DNA repair and/or recombination. Consistent with this, Ku DNA binding activity was shown recently to be absent in extracts of hamster xrs-6 cells, which are defective in DNA double-strand (ds) break repair and V(D)J recombination. Furthermore, xrs-6 cells are complemented by expression of the Ku80 cDNA. To date, DNA-PK activity has been demonstrated unequivocally only in extracts of primate cells. Here, we describe an assay that can detect DNA-PK activity in extracts of mouse, hamster, Xenopus, and Drosophila cells. Using this assay, we find that xrs-6 cells completely lack DNA-PK activity. By contrast, xrs-6 derivatives complemented by human chromosome fragments bearing the Ku80 gene have restored both the DNA end binding and kinase activities associated with DNA-PK. Finally, we show that xrs-6 extracts are complemented biochemically by purified Ku. Our findings indicate that the xrs-6 defects are direct consequences of the mutation in Ku80 and implicate DNA-PK in recombination and DNA repair processes.

Molecular and biochemical characterization of new X-ray-sensitive hamster cell mutants defective in Ku80.

Ku, a heterodimer of approximately 70 and approximately 80 kDa subunits, is a nuclear protein that binds to double-stranded DNA ends and is a component of the DNA-dependent protein kinase (DNA-PK). Cell lines defective in Ku80 belong to group XRCC5 of ionizing radiation-sensitive mutants. Five new independent Chinese hamster cell mutants, XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B, that belong to this group were isolated. To shed light on the nature of the defect in Ku80, the molecular and biochemical characteristics of these mutants were examined. All mutants, except XR-V12B, express Ku80 mRNA, but no Ku80 protein could clearly be detected by immunoblot analysis in any of them. DNA sequence analysis of the Ku80 cDNA from these mutants showed a deletion of 252 bp in XR-V10B; a 6 bp deletion that results in a new amino acid residue at position 107 and the loss of two amino acid residues at positions 108 and 109 in XR-V11B; a missense mutation resulting in a substitution of Cys for Tyr at position 114 in XR-V13B; and two missense mutations in XR-V16B, resulting in a substitution of Met for Val at position 331 and Arg for Gly at position 354. All these mutations cause a similar, 5-7-fold, increase in X-ray sensitivity in comparison to wild-type cells, and a complete lack of DNA-end binding and DNA-PK activities. This indicates that all these mutations lead to loss of the Ku80 function due to instability of the defective protein.

Lack of detectable defect in DNA double-strand break repair and DNA-dependent protein kinase activity in radiosensitive human severe combined immunodeficiency fibroblasts.

The initial step of the V(D)J recombination occurs through the generation of a DNA double-strand break (dsb). Defects in the DNA-dependent protein kinase complex (DNA-PK) result in an inability to perform either V(D)J recombination or any dsb repair effectively. The human autosomal T-B-severe combined immunodeficiency (SCID) condition is characterized by an absence of both B and T lymphocytes and is accompanied in some patients by an increase in gamma-ray sensitivity (T-B-RS SCID) comparable to that found in mouse SCID cells. We show here that cells from six patients with T-B-RS SCID had normal DNA-dsb repair kinetics. Furthermore, DNA-PK activity was present in extracts from these human T-B-RS SCID fibroblasts. We therefore conclude that some human T-B-RS SCID disorders are not caused by a defect in an essential DNA-PK component.

Bacillus anthracis but not always anthrax.

Gram-positive bacilli isolated during epidemiological investigations which, on the basis of conventional tests, resemble Bacillus anthracis but which fail to produce the capsule or to induce anthrax in test animals have long been dismissed in clinical and veterinary laboratories as B. cereus or simply as unidentified Bacillus spp. and thereupon discarded as inconsequential. In this study, the application of newly available DNA probe, polymerase chain reaction and specific toxin antigen detection technology has revealed that a proportion of such strains are B. anthracis which lack the plasmid carrying the capsule gene (pXO2). While these techniques cannot, of course, be used to confirm the identities of strains resembling B. anthracis but which also lack the plasmid carrying the toxin genes (pXO1), the likelihood that these also are bona fide B. anthracis becomes more acceptable. (As yet no naturally occurring pXO1-/2+ strains have been found.) At this point, the significance of the presence of such avirulent forms of B. anthracis in specimens can only be a subject for speculation, but the possibility that they may be indicators of virulent parents somewhere in the system being examined must be considered.

Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation.

Murine cells homozygous for the severe combined immune deficiency mutation (scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PKcs, the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PKcs gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PKcs is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.