Abstract
We examined the mechanism regulating the cellular levels of PNKP, the major kinase/phosphatase involved in the repair of oxidative DNA damage, and find that it is controlled by ATM phosphorylation and ubiquitylation-dependent proteasomal degradation. We discovered that ATM-dependent phosphorylation of PNKP at serines 114 and 126 in response to oxidative DNA damage inhibits ubiquitylation-dependent proteasomal degradation of PNKP, and consequently increases PNKP stability that is required for DNA repair. We have also purified a novel Cul4A-DDB1 ubiquitin ligase complex responsible for PNKP ubiquitylation and identify serine-threonine kinase receptor associated protein (STRAP) as the adaptor protein that provides specificity of the complex to PNKP. Strap(-/-) mouse embryonic fibroblasts subsequently contain elevated cellular levels of PNKP, and show elevated resistance to oxidative DNA damage. These data demonstrate an important role for ATM and the Cul4A-DDB1-STRAP ubiquitin ligase in the regulation of the cellular levels of PNKP, and consequently in the repair of oxidative DNA damage.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Animals
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Ataxia Telangiectasia Mutated Proteins
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Carrier Proteins / metabolism
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Cell Cycle Proteins / metabolism*
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Cullin Proteins / metabolism
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DNA Damage
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DNA Repair Enzymes / chemistry
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DNA Repair Enzymes / metabolism*
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DNA-Binding Proteins / metabolism*
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Enzyme Stability
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HeLa Cells
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Humans
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Mice
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Oxidative Stress*
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Phosphorylation
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Phosphotransferases (Alcohol Group Acceptor) / chemistry
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Phosphotransferases (Alcohol Group Acceptor) / metabolism*
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Proteasome Endopeptidase Complex / metabolism*
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Protein Serine-Threonine Kinases / metabolism*
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Tumor Suppressor Proteins / metabolism*
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Ubiquitin-Protein Ligases / isolation & purification
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Ubiquitin-Protein Ligases / metabolism
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Ubiquitination*
Substances
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CUL4A protein, human
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Carrier Proteins
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Cell Cycle Proteins
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Cullin Proteins
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DDB1 protein, human
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DNA-Binding Proteins
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Strap protein
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Tumor Suppressor Proteins
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Ubiquitin-Protein Ligases
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PNKP protein, human
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Phosphotransferases (Alcohol Group Acceptor)
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Pnkp protein, mouse
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ATM protein, human
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Ataxia Telangiectasia Mutated Proteins
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Atm protein, mouse
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Protein Serine-Threonine Kinases
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Proteasome Endopeptidase Complex
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DNA Repair Enzymes