DNase I-resistant DNA-dependent protein kinase activity in Xenopus oocytes.

DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase consisting of a catalytic subunit p460 (DNA-PKcs), and a DNA binding component termed Ku. DNA-PK plays a role in transcription, nonhomologous recombination, and DNA repair. Several reports have demonstrated that binding to double-stranded DNA (dsDNA) is required for the activation of DNA-PK. To date, very few reports suggest the possibility that an alternative pathway of DNA-PK activation exists without the requirement of dsDNA. In this study, direct biochemical evidence is presented to support this notion. Here, Xenopus oocytes were used as a model system because they offer the advantage of a manual enucleation process providing an extract that can be termed purely 'cytoplasmic' and the isolated nuclei (germinal vesicles) can be used to make nuclear extracts. Specific antibody-mediated pulled-down DNA-PK activity was assayed in the cytoplasmic extracts to evaluate the enzyme activity in the presence and absence of DNA. DNase I treatment did not affect the DNA-PK activity. Analyses of the association of nicked DNA with the pulled-down DNA-PK by radiolabeling the associated nicked DNA provided evidence that the cytoplasmic DNA-PK is catalytically active in absence of DNA. These results suggest that potential mechanisms occurring outside of the nucleus might activate DNA-PK, and therefore, could reveal novel functions of this enzyme.