title: The DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) Are stimulated by bulky adduct-containing DNA.
abstract: A variety of environmental, carcinogenic, and chemotherapeutic agents form bulky lesions on DNA that activate DNA damage checkpoint signaling pathways in human cells. To identify the mechanisms by which bulky DNA adducts induce damage signaling, we developed an in vitro assay using mammalian cell nuclear extract and plasmid DNA containing bulky adducts formed by N-acetoxy-2-acetylaminofluorene or benzo(a)pyrene diol epoxide. Using this cell-free system together with a variety of pharmacological, genetic, and biochemical approaches, we identified the DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) as bulky DNA damage-stimulated kinases that phosphorylate physiologically important residues on the checkpoint proteins p53, Chk1, and RPA. Consistent with these results, purified DNA-PK and ATM were directly stimulated by bulky adduct-containing DNA and preferentially associated with damaged DNA in vitro. Because the DNA damage response kinase ATM and Rad3-related (ATR) is also stimulated by bulky DNA adducts, we conclude that a common biochemical mechanism exists for activation of DNA-PK, ATM, and ATR by bulky adduct-containing DNA.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Kemp
authors.forename: Laura A
authors.initials: LA
authors.lastname: Lindsey-Boltz
authors.forename: Aziz
authors.initials: A
authors.lastname: Sancar
mesh_terms: D000099:Acetoxyacetylaminofluorene / Q000494:pharmacology; D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D001564:Benzo(a)pyrene / Q000494:pharmacology; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D006224:Cricetinae; D003412:Cricetulus; D018736:DNA Adducts / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology*; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000477:Alkylating Agents; D018797:Cell Cycle Proteins; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D001564:Benzo(a)pyrene; D000099:Acetoxyacetylaminofluorene; D011494:Protein Kinases; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases