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The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit
1Department of Biological Sciences, University of Calgary, 2500 University Drive, N.W., Calgary, Alberta, T2N 1N4, Canada.
Abstract:
The DNA-dependent protein kinase (DNA-PK) requires for activity free ends or other discontinuities in the structure of double strand DNA. In vitro, DNA-PK phosphorylates several transcription factors and other DNA-binding proteins and is thought to function in DNA damage recognition or repair and/or transcription. Here we show that in vitro DNA-PK undergoes autophosphorylation of all three protein subunits (DNA-PKcs, Ku p70 and Ku p80) and that phosphorylation correlates with inactivation of the serine/threonine kinase activity of DNA-PK. Significantly, activity is restored by the addition of purified native DNA-PKcs but not Ku, suggesting that inactivation is due to autophosphorylation of DNA-PKcs. Our data also suggest that autophosphorylation results in dissociation of DNA-PKcs from the Ku-DNA complex. We suggest that autophosphorylation is an important mechanism for the regulation of DNA-PK activity.
Insights
Autophosphorylation inactivates DNA-dependent protein kinase (DNA-PK) by affecting its DNA-PKcs subunit. This process regulates DNA-PK activity and may involve dissociation from Ku-DNA complexes.
Area of Science:
- Molecular Biology
- Biochemistry
- Cellular Biology
Background:
- DNA-dependent protein kinase (DNA-PK) is crucial for DNA double-strand break repair and transcription.
- DNA-PK activity relies on discontinuities in double-stranded DNA, such as free ends.
- DNA-PK phosphorylates various transcription factors and DNA-binding proteins.
Purpose of the Study:
- To investigate the mechanism of DNA-PK inactivation.
- To determine the role of autophosphorylation in DNA-PK regulation.
- To elucidate the interaction between DNA-PK subunits during autophosphorylation.
Main Methods:
- In vitro kinase assays to measure DNA-PK activity.
- Analysis of protein phosphorylation status of DNA-PK subunits (DNA-PKcs, Ku70, Ku80).
- Reconstitution experiments with purified DNA-PK components.
Main Results:
- DNA-PK undergoes autophosphorylation of all three subunits (DNA-PKcs, Ku70, Ku80) in vitro.
- Autophosphorylation correlates with the inactivation of DNA-PK serine/threonine kinase activity.
- Re-addition of DNA-PKcs, but not Ku, restored kinase activity, indicating DNA-PKcs autophosphorylation causes inactivation.
- Autophosphorylation leads to the dissociation of DNA-PKcs from the Ku-DNA complex.
Conclusions:
- Autophosphorylation of DNA-PKcs is a key mechanism for regulating DNA-PK activity.
- This autophosphorylation leads to enzyme inactivation and dissociation from DNA-binding complexes.
- Understanding DNA-PK regulation is vital for comprehending DNA repair and transcription pathways.