Formation and fate of cross-links induced by polyfunctional anticancer drugs in yeast

Abstract

A method to detect low levels of interstrand cross-links in DNA of Saccharomyces cerevisiae is described. Isopycnic ultracentrifugation of alkali-treated, unpurified Eaton press homogenates allows the detection of less than one cross-link per yeast chromosome. Efficient separation of single-and double-stranded DNA requires low cell density and addition of glycerol during homogenization. Using a yeast strain defective in excision repair, a dose dependent formation of interstrand cross-links after treatment of cells with biological doses of nitrogen mustard. Triaziquone and Chloramubil could be demonstrated. The most powerful of these alkylating agents is Triaziquone: half of the DNA molecules are shown to be cross-linked after a 12 min exposure to 9×10^-9 g/ml of the drug. The cross-linking reaction continues after excessive alkylating agent is removed. After having reached a maximum the fraction continues after excessive alkylating agent is removed. After having reached a maximum the fraction of renaturable DNA decreases upon further incubation. The speed of this “after-reaction” depends on temperature: 48 h after the end of treatment renaturability of DNA has almost completely disappeared when cells are kept at 36° C.