Kinetic evidence for redistribution of actinomycin molecules between potential DNA-binding sites.

The kinetics of interaction between actinomycin D and DNA have been measured by stopped-flow and detergent-dissociation methods. The results are consistent with a model in which the antibiotic initially binds to many sequences on the heterogeneous DNA lattice and subsequently 'shuffles' between the available sites until a thermodynamically determined optimal state of binding is attained. The amplitudes of the two slowest components in the reaction with calf thymus DNA do not vary in parallel as the total level of antibiotic binding is increased; they appear to reflect directly the redistribution of antibiotic molecules along the DNA lattice. The dissociation profile is shown to depend upon the time for which the antibiotic and DNA are premixed, so that for short mixing times a higher proportion of the decay is represented by faster-dissociating species. The rate of appearance of the slowest-dissociating species correlates well with the slowest optical change in the association reaction. Stopped-flow experiments indicate that the antibiotic first binds to sites on natural DNA with an average association constant of 4 X 10(3) M-1 and that it subsequently migrates to sites with higher affinity. Similar experiments performed with poly(dG-dC) are less easily interpreted and seem to indicate that conformational changes or cooperative effects can also occur.