The effect of the 3′,5′ thiophosphoryl linkage on the exonuclease activities of T4 polymerase and the Klenow fragment

Abstract

The 3′→5′ exonuolease activities of T4 DNA polymerase and the Klenow fragrent of Polymerase I towards the phosphoryl and thiophosphoryl 3′,5′ linkage were examined under conparable conditions of idling-turnover, duplex hydrolysis and turnover during polymerization. with the T4 enzyme there is a negligible, effect of thiosubstitution on these activities; with the Klenow fragment there is a greater than one hundred-fold reduction in rate with the thiolinkage for the exonuclease but not polymerization activities, This inability to hydrolyze rapidly the thiopbospboryl linkage extends to the hydrolytic activity of Exonuolsase III. The quantitstion of the exonuclease activities of these three proteins under various conditions should aid in the successful employment of thiophosphoryl nucleoside triphosphates for their incorporation into DNA.