Freeman-Wittig M J, Lewis R A
Biochim Biophys Acta. 1986 Jun 20;867(3):107-13. doi: 10.1016/0167-4781(86)90070-9.
DNA polymerase I is a multifaceted enzyme with one polymerizing and two exonuclease activities. Captan was previously shown to be an inhibitor of this enzyme's polymerizing activity and this report measures the effects of captan on the two exonuclease activities. When the holoenzyme was tested, captan enhanced the degradation of poly(dA-dT), T7 DNA and, to a significantly lesser extent, heat-denatured DNA. However, when the effects of captan were tested as a function of substrate concentration, the stimulatory influence was measured only at high substrate concentrations. At low concentrations of DNA, captan was inhibitory. Inhibition and enhancement each showed an ED50 of the same value (approx. 100 microM). By assaying the two exonuclease activities separately it was shown that the differential effect on the holoenzyme by captan was the result of a combined inhibition of the 3'----5' exonuclease and enhancement of the 5'----3' exonuclease. Klenow fragment with poly(dA-dT) as substrate was used to assay for 3'----5' exonuclease activity. Captan inhibited this exonuclease and the inhibition could be prevented by the addition of greater concentrations of substrate. Holoenzyme and poly(rA)-poly(dT) were used to assay for 5'----3' exonucleolysis, which was enhanced at higher concentrations of substrate in the presence of captan.
DNA聚合酶I是一种具有多种功能的酶，具有一种聚合活性和两种核酸外切酶活性。之前已证明克菌丹是这种酶聚合活性的抑制剂，本报告测定了克菌丹对两种核酸外切酶活性的影响。当对全酶进行测试时，克菌丹增强了聚（dA-dT）、T7 DNA的降解，对热变性DNA的降解增强程度明显较小。然而，当测试克菌丹的作用与底物浓度的关系时，仅在高底物浓度下才检测到刺激作用。在低DNA浓度下，克菌丹具有抑制作用。抑制和增强作用的半数有效剂量（ED50）值相同（约100 microM）。通过分别检测两种核酸外切酶活性表明，克菌丹对全酶的不同影响是3'→5'核酸外切酶受到抑制以及5'→3'核酸外切酶受到增强的综合结果。以聚（dA-dT）为底物，用Klenow片段检测3'→5'核酸外切酶活性。克菌丹抑制这种核酸外切酶，添加更高浓度的底物可防止这种抑制作用。用全酶和聚（rA）-聚（dT）检测5'→3'核酸外切酶活性，在克菌丹存在下，较高底物浓度时该活性增强。
