Mosbaugh D W
Clayton Foundation Biochemical Institute, University of Texas, Austin 78712.
Nucleic Acids Res. 1988 Jun 24;16(12):5645-59. doi: 10.1093/nar/16.12.5645.
Porcine liver DNA polymerase gamma has been demonstrated to preferentially incorporate dTMP over dUMP during in vitro DNA synthesis. When polymerase activity was measured in standard reactions containing saturating levels of either dTTP or dUTP, the polymerization rate was slightly faster in the reaction containing dTTP. However, under conditions where both dTTP and dUTP competed, at an equal molar concentration, approximately 3-times more thymine residues were incorporated than uracil residues into DNA. Similarly, preferential incorporation of dTMP was observed on several substrates including poly (dA).oligo p(dT), poly (rA).oligo p(dT) and poly (dA-dT). The discrimination against dUMP incorporation was even more apparent with reduced levels of dUTP. These observations were consistent with the finding that the Km for DNA polymerase gamma was about 3-fold lower for dTTP (0.4 microM) than for dUTP (1.1 microM). On the other hand, the Vmax for these two reactions was very similar. Discrimination against dUMP incorporation was also observed during inhibition of polymerase gamma by dideoxyribonucleoside triphosphates. Dideoxythymidine triphosphate preferentially inhibited dUMP incorporation compared to that of dTMP, whereas ddATP, ddCTP and ddGTP inhibited both reactions equally.
猪肝脏DNA聚合酶γ已被证明在体外DNA合成过程中优先掺入dTMP而非dUMP。当在含有饱和水平的dTTP或dUTP的标准反应中测量聚合酶活性时，含有dTTP的反应中聚合速率略快。然而，在dTTP和dUTP相互竞争的条件下，等摩尔浓度时，掺入DNA中的胸腺嘧啶残基比尿嘧啶残基多约3倍。同样，在包括聚（dA）·寡聚p（dT）、聚（rA）·寡聚p（dT）和聚（dA-dT）在内的几种底物上也观察到dTMP的优先掺入。随着dUTP水平降低，对dUMP掺入的歧视更加明显。这些观察结果与以下发现一致，即DNA聚合酶γ对dTTP（0.4 microM）的Km比对dUTP（1.1 microM）低约3倍。另一方面，这两个反应的Vmax非常相似。在双脱氧核糖核苷三磷酸抑制聚合酶γ的过程中也观察到对dUMP掺入的歧视。与dTMP相比，双脱氧胸苷三磷酸优先抑制dUMP掺入，而ddATP、ddCTP和ddGTP对这两个反应的抑制作用相同。
