Proteolytic cleavage fo native DNA polymerase into two different catalytic fragments. Influence of assay condtions on the change of exonuclease activity and polymerase activity accompanying cleavage.

Abstract

Treatment of native DNA polymerase from Escherichia coli with subtilisin may lead to its cleavage into a polymerase fragment and an exonuclease fragment. The cleavage has been followed by electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulfate and by the change both in polymerase activity and in exonuclease activity. The electrophoretic analysis has shown that subtilisin treatment leads to the formation of two new protein bands and simultaneous disappearance of the band corresponding to the native enzyme. The cleavage of the native polymerase may be accompanied by change in the polymerase activity. The change appears to depend on the conditions of the polymerase assay. With activated calf thymus DNA the activity increases by a factor of up to 2.3 when assayed at low phosphate buffer concentration. At higher buffer concentration in the assay mixture the cleavage gives rise to a decrease in polymerase activity. With poly[d(A‐T)] · poly[d(A‐T) · the cleavage gives rise to a decrease in polymerase activity at both low and high buffer concentration. The effect of buffer concentration on the polymerase activity of the native and the cleaved enzyme showed that maximum activity for the two enzymes was obtained at different buffer concentrations in the presence of activated calf thymus DNA. In the presence of poly[d(A‐T)] · poly[d(A‐T)] maximum activity was obtained at the same buffer concentration but the activity of the cleaved enzyme was lower than that of the native at all buffer concentrations. A similar differential effect of ionic strength on the activity has been found for the exonuclease site of the native enzyme and the cleaved exonuclease fragment. This differential effect depends, however, on the molecular size of the substrate poly[d(A‐T)] · poly[d(A‐T)]. It appears, furthermore, that the exonuclease site of the cleaved exonuclease fragment has a smaller affinity towards poly[d(A‐T)] · poly[d(A‐T)] than that of the native enzyme. Finally, while the exonuclease activity of the native enzyme towards poly[d(A‐T)] · poly[d(A‐T)] is stimulated by conditions that permit simultaneous synthesis no such synthesis stimulated exonuclease activity is observed after cleavage of the native enzyme into a polymerase fragment and an exonuclease fragment.