Formation and fate of cross-links induced by polyfunctional anticancer drugs in yeast.
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 Triziquone: half of the DNA molecules are shown to be cross-linked after a 12 min exposure to 9 X 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 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 degrees C.
D000478:Alkylation; D002699:Chlorambucil / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D004247:DNA; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D007700:Kinetics; D008466:Mechlorethamine / Q000494:pharmacology*; D009695:Nucleic Acid Renaturation; D012441:Saccharomyces cerevisiae / Q000187:drug effects*; D014228:Triaziquone / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D002699:Chlorambucil; D008466:Mechlorethamine; D004247:DNA; D014228:Triaziquone