BINDING OF 2,7-DIAMINOMITOSENE TO DNA - MODEL FOR THE PRECOVALENT RECOGNITION OF DNA BY ACTIVATED MITOMYCIN-C

Mitomycin C (MC), mitomycin A, porfiromycin, BMY-25067, and BMY-25287, antitumor antibiotics collectively termed ''mitosanes'', were found to have no appreciable binding affinity to various natural and synthetic DNAs, as tested by UV spectrophotometry and equilibrium dialysis. Further tests of DNA binding applied to MC including thermal melting measurements, displacement of ethidium fluorescence, and unwinding of closed circular DNA were similarly negative. In contrast, 2,7-diaminomitosene (2,7-DAM), a major end product of the reductive activation of MC, binds to the same series of DNAs by all of these criteria. In the presence of DNA its UV absorbance at the 313 nm maximum decreased and underwent a slight red shift. This effect was used for determining DNA binding constants (K-b) by the spectrophotometric titration method. At pH 6.0 the K(b)s of three natural DNAs with varying GC content, as well as poly(dA-dT). poly(dA-dT), and poly(dG-dC). poly(dG-dC), were all in the range of (1.2-5.3) x 10(4) (M nucleotide)(-1), with no apparent specificity of binding. Poly(dG-m5dC). poly(dG-m5dC) displayed a slightly higher K-b ((7.5-8.4) x 10(4)). Binding of other, closely related mitosenes was tested to calf thymus DNA by equilibrium dialysis. Neither the presence of a 1-OH substituent, removal of the 10-carbamoyl group, nor methylation of the 2-amino group modifies the binding affinity of the mitosenes significantly. The 1-phosphate substituent abolishes binding. The binding of 2,7-DAM to DNA increased with decreasing pH and decreasing ionic strength. It was determined that 2,7-DAM is protonated at the 2-amino group with a pK(a) = 7.55, and this correlated well with the observed pH dependence of the binding, indicating that the binding affinity has a strong electrostatic component. This was confirmed by the finding that the extrapolated K-b to 1 M Na+ concentration diminishes to only 10% of the value of K-b at 0.01 M Na+ concentration. Viscosity tests showed conclusively that 2,7-DAM intercalates in DNA, in a nonspecific manner. DNA binding by 2,7-DAM is shown to be a close model of the binding of the reduced activated form of MC, previously characterized indirectly [Teng, S. P., Woodson, S. A., and Crothers, D. M. (1989) Biochemistry 28, 3901-3907]. The nonspecific precovalent binding of the active form may serve in the cell to concentrate the drug at its critical target, DNA. A CpG-specific minor groove binding mode, previously suggested to explain the observed CpG specificity of the covalent alkylation [Kumar, S., Lipman, R., and Tomasz, M. (1992) Biochemistry 31, 1399-1407], is presumably masked by the stronger, bulk nonspecific binding described here.