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 cispl atin-damaged DNA, One protein termed DRP-1 has been purified to homoge neity 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, Th e 83- and 68-kDa subunits in each complex are identical, The 83-kDa su bunit 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 crossreacted with monoclonal antisera raised against the Ku auto antigen p70 subunit, The 350-kDa subunit was identified as DNA-PKcs, t he catalytic subunit of the human DNA-activated protein kinase, DNA-PK , DRP-1/Ku DNA binding was assessed in mobility shift assays and compe tition binding assays using cisplatin-damaged DNA, Results indicate th at DNA binding was essentially unaffected by cisplatin-DNA adducts in the presence or absence of DNA-PKcs, DNA-PK activity was only stimulat ed with undamaged DNA, despite the ability of Ku to bind to cisplatin- damaged DNA. The lack of DNA-PR stimulation by cisplatin-damaged DNA c orrelated with the extent of cisplatin-DNA adduct formation, These res ults demonstrate that Ku can bind cisplatin-damaged DNA but fails to a ctivate DNA-PR, These results are discussed with respect to the repair of cisplatin-DNA adducts and the role of DNA-PK in coordinating DNA r epair processes.