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Nuclear extracts lacking DNA-dependent protein kinase are deficient in multiple round transcription
R L Woodard1, M G Anderson, W S Dynan
1Institute of Molecular Medicine and Genetics, Program in Gene Regulation, Medical College of Georgia, Augusta, Georgia 30912, USA.
Abstract:
We have compared levels of in vitro transcription in nuclear extracts from DNA-dependent protein kinase (DNA-PK)-deficient and DNA-PK-containing Chinese hamster ovary cell lines. DNA-PK-deficient cell lines are radiosensitive mutants lacking either the catalytic subunit or the 80-kDa subunit of the Ku protein regulatory component. Extracts from DNA-PK-deficient cell lines had a 2-7-fold decrease in the level of in vitro transcription when compared with matched controls. This decrease was observed with several promoters. Transcription could be restored to either of the deficient extracts by addition of small amounts of extract from the DNA-PK-containing cell lines. Transcription was not restored by addition of purified DNA-PK catalytic subunit, Ku protein, or individually purified general transcription factors. We conclude that extracts from DNA-PK-deficient cells lack a positively acting regulatory factor or a complex of factors not readily reconstituted with individual proteins. We have also investigated the mechanistic defect in the deficient extracts and have found that the observed differences in transcription levels between Ku-positive and Ku-negative cell lines can be attributed solely to a greater ability of the Ku-positive nuclear extracts to carry out secondary initiation events subsequent to the first round of transcription.
Insights
DNA-dependent protein kinase (DNA-PK)-deficient cells show reduced in vitro transcription due to a lack of regulatory factors. This defect impacts secondary transcription initiation, highlighting DNA-PK
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- DNA-dependent protein kinase (DNA-PK) is crucial for DNA repair and gene regulation.
- Chinese hamster ovary (CHO) cell lines deficient in DNA-PK subunits (catalytic or Ku 80 kDa) exhibit radiosensitivity.
- Understanding DNA-PK's role in transcription is vital for comprehending cellular responses to DNA damage.
Purpose of the Study:
- To compare in vitro transcription levels in DNA-PK-deficient and DNA-PK-containing CHO cell lines.
- To identify the molecular defect responsible for altered transcription in DNA-PK-deficient cells.
- To elucidate the role of DNA-PK and associated factors in regulating gene transcription.
Main Methods:
- In vitro transcription assays using nuclear extracts from DNA-PK-deficient and control CHO cell lines.
- Comparison of transcription levels across various promoters.
- Complementation assays by adding extracts or purified components to deficient extracts.
- Analysis of transcription initiation events, including secondary initiation.
Main Results:
- DNA-PK-deficient cell extracts showed a 2-7 fold decrease in in vitro transcription across multiple promoters.
- Transcription could be restored in deficient extracts by adding whole cell extracts from DNA-PK-containing cells.
- Purified DNA-PK catalytic subunit, Ku protein, or individual transcription factors did not restore transcription.
- Deficient extracts exhibited a reduced capacity for secondary transcription initiation events.
Conclusions:
- DNA-PK-deficient cells lack essential regulatory factors or complexes required for robust transcription.
- The observed transcription defect is linked to impaired secondary initiation events.
- These findings suggest a broader role for DNA-PK-associated factors in regulating transcription beyond DNA repair.