Echinomycin, a bis-intercalating agent, induces C→T mutations via cytosine deamination

Echinomycin, a bis-intercalating, antitumor drug, has been studied for its ability to induce the deamination of cytosine to uracil (C→U) in double-stranded DNA. We have employed a sensitive lacZ α-complementation reversion assay to detect G·C→A·T mutations at a number of sites in M13mp2 DNA to determine the extent to which distortions of DNA structure induced by echinomycin may effect C→U rates. When double-stranded M13mp2 DNA with a 12-base target containing a CpG site was incubated at 37°C, the reversion frequency of the echinomycin-treated DNA increased linearly over time, with a rate constant 3-fold greater than DNA incubated without echinomycin. Of the 11 ways that blue pseudo-revertants can occur in the target, 96% of the observed revertants arose from C→T and tandem CC→TT transitions, with 78% attributable to single-base C→T changes at three sites. Transfection into ung+ cells decreased the reversion frequencies by 85% to near background levels, indicating that the increase in C→T mutations was due to deamination of C to U. The cytosine deamination rate constants for the entire target at pH 6.0 and 37°C were 1.2 × 10−11 sec−1 for untreated DNA and 3.5 × 10−11 sec−1 for echinomycin-treated DNA. The increase in C→T mutation rates occurred at cytosines both proximal and distal to a CpG echinomycin-binding site. We hypothesize that this increase in deamination rate is due to a more open or single-stranded DNA structure caused by the echinomycin: DNA interaction.