CARBOHYDRATE MINOR-GROOVE INTERACTIONS IN THE BINDING OF CALICHEAMICIN GAMMA-1(I) TO DUPLEX DNA

The sequence-specific DNA cleavage activity of calicheamicin gamma(1)( I) was studied using a synthetic 20mer DNA substrate that contains a s ingle TCCT.AGGA target site. The cleavage reaction was initiated by ad dition of beta-mercaptoethanol, which, under conditions of calicheamic in excess, results in burst kinetics. The burst amplitude was used to obtain a K-D value of 135 nM for the calicheamicin-DNA interaction. Ca licheamicin and calicheamicin oligosaccharide were allowed to compete for binding, which provided a K-I value of 4.1 mu M for the oligosacch aride-DNA interaction. Replacement of the iodo-substituted oligosaccha ride by the corresponding bromo-, chloro-, fluoro-, methyl-, and hydro gen-substituted compounds resulted in progressively weaker binding. Re placement of guanine by inosine at the 5'-most but not the 3'-most pos ition within the AGGA target sequence resulted in greatly diminished D NA cleavage. These results suggest that for this target sequence there is a critical interaction between the iodine substituent on the calic heamicin oligosaccharide and the 5'-most guanine C2-NH2 group within t he minor groove of the target DNA.