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.
Kanungo
Jyotshnabala
J
Medical College of Georgia, Institute of Molecular Medicine and Genetics, Augusta 30912, USA. kanungoj@mail.nih.gov
D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D020134:Catalytic Domain; D002457:Cell Extracts; D002467:Cell Nucleus / Q000201:enzymology; D003429:Cross Reactions; D004247:DNA / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004800:Enzymes, Immobilized; D005260:Female; D047468:Immunoprecipitation; D000072200:Ku Autoantigen; D008863:Microspheres; D009865:Oocytes / Q000201:enzymology*; D010455:Peptides / Q000378:metabolism; D012685:Sepharose; D013205:Staphylococcal Protein A / Q000378:metabolism; D014981:Xenopus
D034961:Antigens, Nuclear; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D004800:Enzymes, Immobilized; D010455:Peptides; D013205:Staphylococcal Protein A; D004247:DNA; D012685:Sepharose; D051747:DNA-Activated Protein Kinase; D003850:Deoxyribonuclease I; D000072200:Ku Autoantigen