We have determined the mechanism of DNA-dependent protein kinase (DNA-PK) inhibition by cis-diamminedichloroplatinum(II)- (cisplatin-) damaged DNA. We previously have demonstrated that Ku, the DNA binding subunit of DNA-PK, is capable of binding to DNA duplexes globally damaged with cisplatin but was unable to stimulate DNA-PK(cs), the catalytic subunit [Turchi and Henkels (1996) J. Biol. Chem. 271, 2992-3000]. In this report we have assessed Ku binding and DNA-PK stimulation using a series of DNA substrates containing single, site-specific d(GpG), d(ApG), and d(GpXpG) intrastrand cisplatin adducts and a substrate with a single interstrand cisplatin adduct. Results demonstrate that Ku binding is marginally decreased by the presence of cisplatin adducts on each substrate. When assayed for the ability to stimulate DNA-PK, each cisplatin-damaged substrate resulted in significantly decreased activity compared to undamaged DNA controls. The degree of inhibition of both Ku binding and kinase activity varied depending on the specific adduct employed. The inhibition of DNA-PK activity by cisplatin-damaged DNA was observed using either a synthetic peptide or human replication protein A as a substrate. Autophosphorylation of the DNA-PK(cs) and Ku subunits was also inhibited in reactions performed with cisplatin-damaged DNA, demonstrating that increased autophosphorylation of DNA- PK(cs) does not account for the decreased kinase activity observed with cisplatin-damaged DNA. Equilibrium binding and initial velocity experiments revealed a less than 2-fold increase in the K(d) of Ku and the K(m) of DNA-PK for DNA containing a single 1,2- d(GpG) cisplatin adduct. The mechanism of DNA-PK inhibition by cisplatin-damaged DNA can be attributed to a large decrease in the V(max) and small increase in K(m).
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