Lasken R S, Schuster D M, Rashtchian A
Life Technologies, Inc., Gaithersburg, Maryland 20884-9980, USA.
J Biol Chem. 1996 Jul 26;271(30):17692-6. doi: 10.1074/jbc.271.30.17692.
We show that archaebacterial DNA polymerases are strongly inhibited by the presence of small amounts of uracil-containing DNA. Inhibition appears to be competitive, with the DNA polymerase exhibiting approximately 6500-fold greater affinity for binding the inhibitor than a DNase I-activated DNA substrate. All six archaebacterial DNA polymerases tested were inhibited, while no eubacterial, eukaryotic, or bacteriophage enzymes showed this effect. Only a small inhibition resulted when uracil was present as the deoxynucleoside triphosphate, dUTP. The rate of DNA synthesis was reduced by approximately 40% when dUTP was used in place of dTTP for archaebacterial DNA polymerases. Furthermore, an incorporated dUMP served as a productive 3'-primer terminus for subsequent elongation. In contrast, the presence of an oligonucleotide containing as little as a single dUrd residue was extremely inhibitory to DNA polymerase activity on other primer-template DNA.
我们发现，少量含尿嘧啶的DNA的存在会强烈抑制古细菌DNA聚合酶。这种抑制似乎是竞争性的，与DNA底物相比，DNA聚合酶对抑制剂的结合亲和力大约高6500倍。所测试的六种古细菌DNA聚合酶均受到抑制，而真细菌、真核生物或噬菌体的酶均未表现出这种效应。当尿嘧啶以脱氧核苷三磷酸dUTP的形式存在时，仅产生轻微抑制。当用dUTP代替dTTP用于古细菌DNA聚合酶时，DNA合成速率降低了约40%。此外，掺入的dUMP作为后续延伸的有效3'-引物末端。相反，含有低至单个dUrd残基的寡核苷酸的存在对其他引物-模板DNA上的DNA聚合酶活性具有极强的抑制作用。
