Human Ku autoantigen binds cisplatin-damaged DNA but fails to stimulate human DNA-activated protein kinase.
We have identified a series of proteins based on an affinity for cisplatin-damaged DNA. One protein termed DRP-1 has been purified to homogeneity and was isolated as two distinct complexes. The first complex is a heterodimer of 83- and 68-kDa subunits, while the second complex is a heterotrimer of 350-, 83-, and 68-kDa subunits in a 1:1:1 ratio. The 83- and 68-kDa subunits in each complex are identical. The 83-kDa subunit of DRP-1 was identified as the p80 subunit of Ku autoantigen by N-terminal protein sequence analysis and reactivity with a monoclonal antibody directed against human Ku p80 subunit. The 68-kDa subunit of DRP-1 cross-reacted with monoclonal antisera raised against the Ku autoantigen p70 subunit. The 350-kDa subunit was identified as DNA-PKcs, the catalytic subunit of the human DNA-activated protein kinase, DNA-PK. DRP-1/Ku DNA binding was assessed in mobility shift assays and competition binding assays using cisplatin-damaged DNA. Results indicate that DNA binding was essentially unaffected by cisplatin-DNA adducts in the presence or absence of DNA-PKcs. DNA-PK activity was only stimulated with undamaged DNA, despite the ability of Ku to bind to cisplatin-damaged DNA. The lack of DNA-PK stimulation by cisplatin-damaged DNA correlated with the extent of cisplatin-DNA adduct formation. These results demonstrate that Ku can bind cisplatin-damaged DNA but fails to activate DNA-PK. These results are discussed with respect to the repair of cisplatin-DNA adducts and the role of DNA-PK in coordinating DNA repair processes.
Turchi
J J
JJ
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA.
Henkels
K
K
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D001324:Autoantigens / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002945:Cisplatin / Q000378:metabolism / Q000633:toxicity*; D003429:Cross Reactions; D004247:DNA / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D001324:Autoantigens; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C057270:cisplatin-DNA adduct; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D002945:Cisplatin