DNA Polymerase III of B.subtilis: Characterization of the Binding Site for Arylhydrazinopyrimidine Inhibitors

Abstract

A distinct class of 6-substituted pyrimidines, exemplified by the 6-(arylazo)pyrimidine, 6-(p-hydroxyphenylazo)uracil (HPUra)) selectively inhibits the replicative synthesis of DNA of Gram-positive bacteria (1). The site of drug action is the replication-specific enzyme, DNA polymerase III (2,3,4) (Pol III). The mechanism of pol III inhibition by HPUra and analogous 6-substituted uracils — a mechanism which has been studied in detail in our laboratory and that of Cozzarelli (3,4,5,6,7,8,9) — involves the following three processes:- (1) reduction of the drug to an active, hydrazino (H₂) form (i.e.,$-N=N-\to -\begin{matrix} N \\ H \\ \end{matrix}-\begin{matrix} N \\ H \\ \end{matrix}-$, (2) hydrogen bonding, in competition with dGTP, to an unpaired cytosine residue in the template strand immediately distal to the 3’ hydroxyl primer terminus, i.e. 3’OH and, (3) reversible reaction of the template-bound drug with enzyme, sequestering the latter from productive activity as a component of a relatively stable, ternary complex. The novel mechanism by which H₂·HPUra pairs with cytosine (4) and our current concept of the drug:DNA:enzyme complex (8) are depicted, respectively, in parts A and B of Fig. 1.

A.S.I. Participant