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 (rb) 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 rb = 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.
ASJC Scopus subject areas