Nevinskiĭ G A, Doronin S V, Podust V N, Lavrik O I
Mol Biol (Mosk). 1987 Jul-Aug;21(4):1070-9.
The modification of the human placenta DNA polymerase alpha by the imidazolides of dNMP was investigated. The modification was shown to occur only in the simultaneous presence of the template and the primer. This process, however, doesn't depend on the complementary interaction of the nucleotide base with the template. The Kd values of the complexes between the different nucleotides and DNA polymerase alpha were estimated. The affinity of Im-dTMP was determined from the dependence of the Kapp of the enzyme inactivation rate on the reagent concentration. The Kd values for dNMP, dNDP, dNTP were estimated using the protective effect of these nucleotides under the enzyme modification by Im-dTMP. The comparison of the interaction efficiency between the polymerase and dNMP, dNDP, dNTP (complementary or non-complementary to the template) allow to conclude that the nucleotide discrimination occurs on the dNTP level, i. e. dNMP and dNDP upon forming the complex with the enzyme, don't interact complementarily with the template. The additional contacts between the enzyme and the nucleotide terminal phosphate were supposed to form only for the complementary dNTP. The studies allowed to put forward a hypothetical model of the template complementary dNTP binding to the polymerases. The role of the hydrophobic interaction of the nucleotides with the enzyme as well as the possible influence of the nucleotide gamma-phosphate group on the template--dNTP complement formation. The Watson-Crick bound formation of the nucleotide with the template was supposed to be followed by the additional conformational rearrangement of the nucleotide triphosphate chain. The latter process leads to the formation of additional contacts between the enzyme and the nucleotide gamma-phosphate.
研究了dNMP的咪唑化物对人胎盘DNA聚合酶α的修饰作用。结果表明，这种修饰仅在模板和引物同时存在时发生。然而，这一过程并不依赖于核苷酸碱基与模板的互补相互作用。估算了不同核苷酸与DNA聚合酶α之间复合物的Kd值。根据酶失活速率的表观解离常数（Kapp）对试剂浓度的依赖性确定了Im-dTMP的亲和力。利用这些核苷酸在Im-dTMP对酶的修饰作用下的保护效应，估算了dNMP、dNDP、dNTP的Kd值。比较聚合酶与dNMP、dNDP、dNTP（与模板互补或非互补）之间的相互作用效率，可以得出核苷酸识别发生在dNTP水平上的结论，即dNMP和dNDP与酶形成复合物时，不会与模板进行互补相互作用。推测酶与核苷酸末端磷酸之间的额外接触仅为互补dNTP所形成。这些研究提出了模板互补dNTP与聚合酶结合的假设模型。研究了核苷酸与酶的疏水相互作用的作用以及核苷酸γ-磷酸基团对模板-dNTP互补形成的可能影响。核苷酸与模板的沃森-克里克结合形成之后，预计三磷酸核苷酸链会发生额外的构象重排。后一过程导致酶与核苷酸γ-磷酸之间形成额外的接触。
