Influence of polyamines on the activity of DNA polymerase I from Escherichia coli

Abstract

The influence of polyamines on the various activities of DNA polymerase I from Escherichia coli (EC 2.7.7.7) has been investigated. For all high molecular weight DNAs spermine and spermidine caused up to 80% inhibition when present in high concentrations, i.e. above 1 mM for spermine and 2 mM for spermidine. In the presence of low concentrations of polyamines a small activation was seen for some DNAs. The diamines cadaverine and putrescine had little influence on the rate of synthesis with natural occurring DNAs. In the case of d(A—T) n the activation/inhibition was found to be markedly dependent on the molecular weight of the samples used. With a low molecular weight DNA, 5.6 S, addition of spermidine resulted in up to 3-fold stimulation of activity. The activation was dependent on the concentration of MgCl 2 and ionic strength; increasing concentration of these gave a decrease in the degree of activation. Polyamines also had a dramatic effect on the rate of synthesis using the homopolymers (dA) n · (dT) 10 and (rA) n · (dT) 10 (20 : 1) as primers. Putrescine, in particular, increased the activity up to 10-fold with (rA) n · (dT) 10 and somewhat less for (dA) n · (dT) 10. The apparent K m for the primer (rA) 10 · (dT) 10 decreased approx. 35-fold in the presence of 6.6 mM putrescine. There was no influence on the apparent K m for dTTP. The influence of polyamines on both the 5′ → 3′ and 3′ → 5′ nuclease activity was also investigated. Inhibition of nuclease activity was observed in the presence of polyamines, particularly with spermine. Thus with d(A—T) n and T 7 DNA as substrates addition of 0.7 mM spermine resulted in almost complete inhibition of activity. The dramatic inhibition observed with high concentrations of spermine (spermidine) both in the case of polymerizing and nuclease activity is thought to be due to polyamine-induced aggregation of DNA molecules.