Replicative DNA polymerases are able to discriminate between very similar substrates with high accuracy. One mechanism by which E. coli DNA polymerase I checks for Watson–Crick geometry is through a hydrogen bonding fork between Arg668 and the incoming dNTP and the minor groove of the primer terminus. The importance of the Arg-fork was examined by disrupting it with either a guanine to 3-deazaguanine substitution at the primer terminus or the use of a carbocyclic deoxyribose analog of dUTP. Using thio-substituted dNTPs and differential quench techniques, we determined that when the Arg-fork was disrupted, the rate-limiting step changed from a conformational change to phosphodiester bond formation. This result indicates that Arg668 is involved in the phosphoryl transfer step. We examined the role of the Arg-fork in the replication of four DNA damaged templates, O⁶-methylguanine (O⁶-mG), 8-oxo-7,8-dihydroguanine (oxoG), O²-[4-(3-pyridyl)-4-oxobutyl]thymine (O²-POB-T), and N²-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro-8,9,10-trihydroxybenzo[a]pyren-7-yl]-guanine (N²-BP-G). In general, the guanine to 3-deazaguanine substitution caused a decrease in k_pol that was proportional to k_pol over five orders of magnitude. The linear relationship indicates that the Arg668-fork helps catalyze phosphoryl transfer by the same mechanism with all the substrates. Exceptions to the linear relationship were the incorporations of dTTP opposite G, oxoG, and O⁶mG, which showed large decreases in k_pol, similar to that exhibited by the Watson–Crick base pairs. It was proposed that the incorporation of dTTP opposite G, oxoG, and O⁶mG occurred via Watson–Crick-like structures.

中文翻译：

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活性位点相互作用在大肠杆菌DNA聚合酶I复制受损和未损坏的DNA的过程中影响磷酸转移。

复制性DNA聚合酶能够以很高的准确度区分非常相似的底物。大肠杆菌的一种机制DNA聚合酶I通过Arg668与进入的dNTP和引物末端的小沟之间的氢键叉检查Watson-Crick的几何形状。通过在引物末端用鸟嘌呤取代3-脱氮鸟嘌呤或使用dUTP的碳环脱氧核糖类似物破坏Arg-fork的重要性进行了研究。使用硫取代的dNTPs和差异猝灭技术，我们确定当Arg叉被破坏时，限速步骤从构象变化变为磷酸二酯键形成。该结果表明Arg668参与磷酸基转移步骤。我们研究了Arg叉在四个DNA受损模板O ^6-甲基鸟嘌呤（O ⁶-mG），8-氧代-7,8-二氢鸟嘌呤（oxoG），O ^2- [4-（3-吡啶基）-4-氧代丁基]胸腺嘧啶（O ² -POB-T）和N ² -[（7 S，8 R，9 S，10 R）-7,8,9,10-四氢-8,9,10-三羟基苯并[ a ] py-7-基]-鸟嘌呤（N ² -BP -G）。通常，鸟嘌呤到3-脱氮鸟嘌呤置换引起的降低ķ _POL这是正比于ķ _POL超过五个数量级。线性关系表明，Arg668叉与所有底物的催化机理相同，均有助于催化磷酰基转移。线性关系的例外是与G，oxoG和O ⁶ mG相对的dTTP的掺入，这表明k _pol大大降低，类似于Watson-Crick碱基对所显示的。有人提出，与G，oxoG和O ⁶ mG相反的dTTP的掺入是通过Watson-Crick样结构发生的。