Turchi J J, Patrick S M, Henkels K M
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA.
Biochemistry. 1997 Jun 17;36(24):7586-93. doi: 10.1021/bi963124q.
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-PKcs, the catalytic subunit [Turchi & 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-PKcs and Ku subunits was also inhibited in reactions performed with cisplatin-damaged DNA, demonstrating that increased autophosphorylation of DNA-PKcs 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 Kd of Ku and the Km 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 Vmax and small increase in Km.
我们已经确定了顺二氯二氨铂（II）（顺铂）损伤的DNA抑制DNA依赖性蛋白激酶（DNA-PK）的机制。我们之前已经证明，DNA-PK的DNA结合亚基Ku能够结合被顺铂全局损伤的DNA双链体，但无法刺激催化亚基DNA-PKcs [Turchi & Henkels（1996年）《生物化学杂志》271, 2992 - 3000]。在本报告中，我们使用了一系列含有单个位点特异性d（GpG）、d（ApG）和d（GpXpG）链内顺铂加合物的DNA底物以及一个含有单个链间顺铂加合物的底物，评估了Ku的结合和DNA-PK的刺激作用。结果表明，每个底物上顺铂加合物的存在使Ku的结合略有减少。当检测刺激DNA-PK的能力时，与未损伤的DNA对照相比，每个顺铂损伤的底物导致活性显著降低。Ku结合和激酶活性的抑制程度因所使用的特定加合物而异。使用合成肽或人复制蛋白A作为底物时，观察到顺铂损伤的DNA对DNA-PK活性的抑制作用。在用顺铂损伤的DNA进行的反应中，DNA-PKcs和Ku亚基的自磷酸化也受到抑制，这表明DNA-PKcs自磷酸化的增加并不能解释顺铂损伤的DNA所观察到的激酶活性降低。平衡结合和初始速度实验表明，对于含有单个1,2 - d（GpG）顺铂加合物的DNA，Ku的Kd和DNA-PK的Km增加不到2倍。顺铂损伤的DNA抑制DNA-PK的机制可归因于Vmax的大幅降低和Km的小幅增加。
