On the kinetics of distamycin binding to its target sites on duplex DNA

Distamycin A is a well known polyamide antibiotic that can bind in the mino r groove of duplex DNA primarily at AT-rich sequences both as a monomer or as a side-by-side antiparallel dimer. The association phase of the distamyc in binding reaction has not been studied in either of its binding modes, be cause of the lack of an adequate UV or CD signal at the low concentrations needed to monitor the fast bimolecular reaction. We report a significant in crease in fluorescence amplitude, accompanied by a small red shift, on bind ing distamycin to its specific target sites. This signal can be used to mon itor drug binding in steady-state and time-resolved processes. Distamycin s hows extremely fast association with the 1:1 binding site, with a bimolecul ar rate of 7 x 10(7) M-1.s(-1) and also fairly rapid dissociation (approxim ate to 3 s(-1)). When DNA is in excess, there is a slow component in the as sociation reaction whose rate decreases strongly with increasing DNA concen tration. Binding of the drug to the 2:1 site occurs in two distinct steps: fast, sequential binding of each drug molecule to the DNA with a bimolecula r rate comparable to that at the 1:1 site, followed by a slow (approximate to 4 S-1) equilibration to the final population. Dissociation from the 2:1 site is approximate to 40-fold slower than from the 1:1 site. This study pr ovides the groundwork for analysis of the binding kinetics of longer polyam ides and covalently linked polyamides that have recently been shown to inhi bit transcription in vivo.