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Ku entry into DNA inhibits inward DNA transactions in vitro.
Ku entry into DNA inhibits inward DNA transactions in vitro.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2000 Nov 17; Vol. 275 (46), pp. 35684-91. - Publication Year :
- 2000
-
Abstract
- Association of the DNA end-binding Ku70/Ku80 heterodimer with the 460-kDa serine/threonine kinase catalytic subunit forms the DNA-dependent protein kinase (DNA-PK) that is required for double-strand break repair by non-homologous recombination in mammalian cells. Recently, we have proposed a model in which the kinase activity is required for translocation of the DNA end-binding subunit Ku along the DNA helix when DNA-PK assembles on DNA ends. Here, we have questioned the consequences of Ku entry into DNA on local DNA processes by using human nuclear cell extracts incubated in the presence of linearized plasmid DNA. As two model processes, we have chosen nucleotide excision repair (NER) of UVC DNA lesions and transcription from viral promoters. We show that although NER efficiency is strongly reduced on linear DNA, it can be fully restored in the presence of DNA-PK inhibitors. Simultaneously, the amount of NER proteins bound to the UVC-damaged linear DNA is increased and the amount of Ku bound to the same DNA molecules is decreased. Similarly, the poor transcription efficiency exhibited by viral promoters on linear DNA is enhanced in the presence of DNA-PK inhibitor concentrations that prevent Ku entry into the DNA substrate molecule. The present results show that DNA-PK catalytic activity can regulate DNA transactions including transcription in the vicinity of double-strand breaks by controlling Ku entry into DNA.
- Subjects :
- Androstadienes pharmacology
Base Sequence
DNA chemistry
DNA genetics
DNA Damage
DNA Repair drug effects
DNA, Viral chemistry
DNA, Viral genetics
DNA, Viral metabolism
DNA-Activated Protein Kinase
Enzyme Inhibitors pharmacology
HeLa Cells
Humans
Ku Autoantigen
Molecular Sequence Data
Nucleic Acid Conformation
Plasmids chemistry
Plasmids genetics
Plasmids metabolism
Promoter Regions, Genetic genetics
Protein Binding drug effects
Protein Serine-Threonine Kinases antagonists & inhibitors
Protein Serine-Threonine Kinases genetics
Transcription, Genetic drug effects
Ultraviolet Rays
Wortmannin
Antigens, Nuclear
DNA metabolism
DNA Helicases
DNA-Binding Proteins metabolism
Nuclear Proteins metabolism
Protein Serine-Threonine Kinases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 275
- Issue :
- 46
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 10945984
- Full Text :
- https://doi.org/10.1074/jbc.M004315200