Cooperativity and Competition in the Binding of Heterocyclic Diamidines and RNA Polymerases to phiX174 DNA

Abstract

Previously, restriction enzyme activity assays were employed to locate, separately, binding sites for T7 RNA polymerase, sp6 RNA polymerase and the heterocyclic diamidines DB75, DB293 and DB818 on phiX174 DNA. Frequently, these sites are in similar locations. RNA polymerases have been suggested to move along the DNA molecule in search of binding targets. The presence of a bound heterocyclic diamidine in the vicinity of an RNA polymerase binding site could alter the binding of the polymerase. Restriction enzyme activity assays with ten restriction enzymes possessing different cleavage sequences and different flanking sequences were used to examine the mixed binding of either DB75, DB293, or DB818 with either T7 RNA polymerase or sp6 RNA polymerase to phiX 174 DNA. Studies were done holding [diamidine] constant and varying the [polymerase] and also holding [polymerase] constant with varied [diamidine]. Frequently, the binding of the diamidine and the polymerase were complementary. For example, combining DB 818 and T7 RNA polymerase produced increased inhibition with Bssh II [cleavage site GCGCGC], Dra I [TTTAAA], Mlu I [ACGCGT]. Similar results were found with the combination of DB75 and T7 RNA polymerase and the combination of sp6 polymerase and DB75. In contrast, DB293 and T7 polymerase frequently appeared to work in a contrary fashion or did not alter the effects of the other. With Mlu I, addition of DB293 countered the effect of T7 and addition of T7 countered the effect of DB293. It is possible that DB293 alters the conformation of DNA such that it alters the binding of the polymerase. We previously showed that DB293 binds in a contrary fashion to DB 818. These studies provide further evidence that antiparasitic agents such as these diamidines may function by altering DNA-polymerase activity.