Knorre D G, Lavrik O I, Nevinsky G A
Institute of Bioorganic Chemistry, Siberian Division of the Academy of Sciences, Novosibirsk, U.S.S.R.
Biochimie. 1988 May;70(5):655-61. doi: 10.1016/0300-9084(88)90250-7.
The affinities of oligothymidylates and of some analogs for the template site, of a set of oligodeoxyribo- and oligoribonucleotides for the primer site, and of dNTPs and some analogs for the substrate sites of DNA polymerase I Klenow fragment and of human placenta DNA polymerase alpha were measured using them either as competitors of affinity modification or as substrates. The data obtained enable us to hypothesize that the Me2+-dependent electrostatic contact and hydrogen bond of a single internucleotide phosphate and the hydrophobic interactions of the other nucleotide units determine the formation of oligonucleotide-template site complexes. Interaction of the primer's 3'-terminal hydroxy group and of the negatively charged adjacent phosphate with the enzyme, and Watson-Crick base pairing with the template are of crucial importance for the formation of the ternary enzyme-template-primer complex. dNTP and dNMP imidazolides inactivate enzymes via an affinity modification mechanism only in the presence of the template-primer complex. dNTP affinities exceed those of dNDPs and dNMPs, the enhancement being most significant for the substrate that is complementary to the template, thus suggesting the participation of the gamma-phosphate of dNTP in the substrate selection step.
使用寡聚胸苷酸及其某些类似物作为亲和修饰的竞争者或底物，测定了它们与模板位点的亲和力；使用一组寡脱氧核糖核苷酸和寡核糖核苷酸作为底物，测定了它们与引物位点的亲和力；使用脱氧核苷三磷酸（dNTP）及其某些类似物作为底物，测定了它们与DNA聚合酶I Klenow片段和人胎盘DNA聚合酶α底物位点的亲和力。所获得的数据使我们能够推测，单个核苷酸间磷酸基团的Me2 +依赖性静电接触和氢键以及其他核苷酸单元的疏水相互作用决定了寡核苷酸 - 模板位点复合物的形成。引物3'-末端羟基和带负电荷的相邻磷酸基团与酶的相互作用，以及与模板的沃森 - 克里克碱基配对对于三元酶 - 模板 - 引物复合物的形成至关重要。只有在模板 - 引物复合物存在的情况下，dNTP和dNMP咪唑化物才通过亲和修饰机制使酶失活。dNTP的亲和力超过dNDP和dNMP，对于与模板互补的底物，这种增强最为显著，因此表明dNTP的γ-磷酸基团参与了底物选择步骤。
