Multiple Binding Modes of Actinomycin D Reveal the Basis for its Potent HIV-1 and Cancer Activity

Abstract

Actinomycin D (ActD) is a well studied anti-cancer agent that is used as a prototype for developing new generations of drugs. However, the biophysical basis of its activity is still unclear. Because ActD is known to intercalate double stranded DNA (dsDNA), it was assumed to block replication by stabilizing dsDNA in front of the replication fork. However, recent studies have shown that ActD binds with even higher affinity to imperfect duplexes and some sequences of single stranded DNA (ssDNA). These features suggest that ActD may alternatively destabilize complementary dsDNA. In this work we use optical tweezers to stretch and relax single dsDNA molecules in the presence of varying ActD concentrations. We observe that ActD binds with highest affinity to two separate DNA strands that are connected by ActD. This binding mode is ∼1000-fold stronger than ActD's intercalation into dsDNA. We are able to characterize at least two classes of ActD-ssDNA binding sites that differ in dissociation times (∼10% of sites with ∼1000 sec off time, and the rest with ∼10 sec off time). The much weaker ActD binding to dsDNA relative to ssDNA leads to duplex destabilization, in contrast to conventional intercalation. At saturation, the ActD-dsDNA complex becomes indistinguishable from the saturated ActD-ssDNA. These results suggest that two separate, anti-parallel DNA strands constitute the highest affinity natural substrate for ActD binding, with Kd ∼10-100 nM and a relatively slow off rate. This finding supports the hypothesis that the primary characteristic of ActD that contributes to its biological activity is its ability to inhibit cellular replication by stabilizing DNA bubbles during RNA transcription, thereby stalling the transcription process.