Inhibition of the BamHI Cleavage and Unwinding of pBR322 Deoxyribonucleic Acid by the Antitumor Drug cis-Dichlorodiammineplatinum (II)

The antitumor drug m-dichlorodiammineplatinum(II) (cis-DDP) binds to pBR322 DNA and inhibits the cleavage of this circular DNA into a linear form by the restriction endonuclease BamHI. The binding of platinum to DNA was monitored by agarose gel electrophoresis, and the amount of platinum bound per nucleotide (r_b) was measured by carbon rod atomic absorption spectroscopy. Electrophoretic mobility changes reflect a shortening and unwinding of the DNA duplex upon platinum binding as observed previously for the reaction of cis- and frans-DDP with pSMl DNA [Cohen, G. L., Bauer, W. R., Barton, J. K., & Lippard, S. J. (1979) Science (Washington, D.C.) 203, 1014-1016], The inhibition of BamUl nuclease activity occurs at very low binding levels and is complete at r_b = 0.045. This value corresponds to the binding of one platinum atom within ±3 base pairs of the recognition sequence of the enzyme shown below. Treatment of the DNA with 0.2 M sodium cyanide after BamUl cutting removes the platinum but does not alter the point at which cij-DDP inhibits the formation of the linear form III DNA. This result is in contrast with a previous report claiming that BamUl could cut across a m-DDP-induced GpG cross-link in DNA which could be subsequently revealed by cyanide reversal of platinum binding. When the platinum is removed by cyanide treatment, the drug-induced mobility changes are reversed and there is a pronounced sharpening of the bands in the gel. Quantitative study of the cyanide reversal shows the presence of a small amount of unremovable platinum tightly bound to the DNA at high ratios (~0.1) of bound platinum per nucleotide.