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 t o have no appreciable binding affinity to various natural and syntheti c DNAs, as tested by UV spectrophotometry and equilibrium dialysis. Fu rther tests of DNA binding applied to MC including thermal melting mea surements, displacement of ethidium fluorescence, and unwinding of clo sed circular DNA were similarly negative. In contrast, 2,7-diaminomito sene (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 pre sence of DNA its UV absorbance at the 313 nm maximum decreased and und erwent a slight red shift. This effect was used for determining DNA bi nding constants (K-b) by the spectrophotometric titration method. At p H 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 i n 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 sligh tly 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. Neith er the presence of a 1-OH substituent, removal of the 10-carbamoyl gro up, 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 a nd decreasing ionic strength. It was determined that 2,7-DAM is proton ated at the 2-amino group with a pK(a) = 7.55, and this correlated wel l with the observed pH dependence of the binding, indicating that the binding affinity has a strong electrostatic component. This was confir med 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 clo se model of the binding of the reduced activated form of MC, previousl y 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 d rug at its critical target, DNA. A CpG-specific minor groove binding m ode, previously suggested to explain the observed CpG specificity of t he covalent alkylation [Kumar, S., Lipman, R., and Tomasz, M. (1992) B iochemistry 31, 1399-1407], is presumably masked by the stronger, bulk nonspecific binding described here.