Requirement for the Kinase Activity of Human DNA-Dependent Protein Kinase Catalytic Subunit in DNA Strand Break Rejoining
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- Akihiro Kurimasa
- Life Sciences Division
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- Satoshi Kumano
- Life Sciences Division
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- Nikolai V. Boubnov
- Department of Biochemistry and Molecular Biology, St. Louis University, St. Louis, Missouri 63104
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- Michael D. Story
- Department of Experimental Radiotherapy, M. D. Anderson Cancer Center, University of Texas, Houston, Texas 77030
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- Chang-Shung Tung
- Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
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- Scott R. Peterson
- Life Sciences Division
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- David J. Chen
- Life Sciences Division
説明
The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs.
収録刊行物
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- Molecular and Cellular Biology
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Molecular and Cellular Biology 19 (5), 3877-3884, 1999-05-01
Informa UK Limited
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キーワード
- Models, Molecular
- Recombination, Genetic
- Binding Sites
- DNA Repair
- Genes, Immunoglobulin
- Molecular Sequence Data
- Gene Expression
- Nuclear Proteins
- DNA-Activated Protein Kinase
- Protein Serine-Threonine Kinases
- Cyclic AMP-Dependent Protein Kinases
- Recombinant Proteins
- DNA-Binding Proteins
- Phosphatidylinositol 3-Kinases
- Adenosine Triphosphate
- Tumor Cells, Cultured
- Humans
- Amino Acid Sequence
- Sequence Alignment
- DNA Damage
