Action of intercalating agents on the activity of DNA polymerase I.

Abstract:

The effect of intercalating compounds such as 9-aminoacridine, quinacrine (atebrin), proflavine and daunomycin on the activity of DNA polymerase I(EC 2.7.7.7) was studied in vitro and compared with the binding of these acridines to native DNA. The enzyme kinetics were followed at various concentrations of DNA 3'-OH primer end groups and constant concentrations of deoxynucleosidetriphosphates as well as under the opposite conditions. The Km values for the DNA 3'-OH end groups were 16--38 nM and for the deoxynucleosidetriphosphates 2--5 micrometer, depending on the buffer and pH used in the enzymatic assay. All acridine derivates inhibit the DNA polymerase; at variable DNA concentrations a competitive inhibition was observed, where the Ki values ranged between 0.87 and 8.5 micrometer. At variable concentrations of deoxynucleosidetriphosphates and constant DNA concentration a non-competitive inhibition was observed. On denatured 3'-OH DNA as well as on poly(dA) - (dT)10 as substrate no inhibition by 9-aminoacridine was observed. 5'--3' exonuclease activity of DNA polymerase is inhibited by 9-aminoacridine but 3'--5' exonuclease activity on denatured DNA is not influenced by this intercalating compound. The affinity of the acridines to DNA was determined spectrophotometrically under conditions similar to those in the enzymatic assay and the computed frequency of intercalation was related to the inhibition of enzymatic activity. The mechanism of inhibition is explained by a disturbance of the structure of the double helical DNA due to the interaction of the bound acridine derivates.

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