Department of Pharmacology, Case Western Reserve University,10900 Euclid Avenue, Cleveland, Ohio 44106, USA.
Biochemistry. 2010 Apr 13;49(14):3009-23. doi: 10.1021/bi901523y.
Most models accounting for the efficiency and fidelity of DNA polymerization invoke the use of either hydrogen bonding contacts or complementarity of shape and size between the formed base pair. This report evaluates these mechanisms by quantifying the ability of a high-fidelity DNA polymerase to replicate 5-nitroindole, a purine mimetic devoid of classic hydrogen bonding capabilities. 5-NITP acts as a universal nucleotide since it is incorporated opposite any of the four natural nucleobases with nearly equal efficiencies. Surprising, the polymerization reaction is not reciprocal as natural nucleotides are poorly incorporated opposite 5-nitroindole in the template strand. Incorporation opposite 5-nitroindole is more efficient using natural nucleotides containing various modifications that increase their base stacking potential. However, 5-substituted indolyl nucleotides that contain pi-electron and/or hydrophobic groups are incorporated opposite the non-natural nucleobase with the highest catalytic efficiencies. The collective data set indicate that replication of a non-natural nucleobase is driven by a combination of the hydrophobic nature and pi-electron surface area of the incoming nucleotide. In this mechanism, the overall hydrophobicity of the incoming nucleobase overcomes the lack of hydrogen bonding groups that are generally required for optimal DNA polymerization. However, the lack of hydrogen bonds between base pairs prevents primer extension. This final aspect is manifest by the appearance of unusually high pyrophosphorolysis activity by the T4 DNA polymerase that is only observed with the non-natural nucleobase in the template. These results highlight the importance of hydrogen bonding interactions during primer extension and pyrophosphorolysis.
大多数解释 DNA 聚合效率和保真度的模型都采用氢键接触或形成碱基对的形状和大小互补的机制。本报告通过量化高保真 DNA 聚合酶复制 5-硝基吲哚（一种缺乏经典氢键能力的嘌呤类似物）的能力来评估这些机制。5-NITP 作为一种通用核苷酸，因为它可以与任何四种天然核碱基几乎以相等的效率配对。令人惊讶的是，聚合反应不是相互的，因为天然核苷酸在模板链中与 5-硝基吲哚的配对掺入效率很低。使用各种修饰物增加其碱基堆积潜力的天然核苷酸，其掺入对 5-硝基吲哚的反应更有效。然而，含有π电子和/或疏水性基团的 5-取代吲哚核苷酸与非天然碱基的结合具有最高的催化效率。总的数据集表明，非天然碱基的复制是由进入核苷酸的疏水性和π电子表面积的组合驱动的。在这种机制中，进入碱基的整体疏水性克服了通常需要最佳 DNA 聚合的氢键基团的缺乏。然而，碱基对之间缺乏氢键会阻止引物延伸。T4 DNA 聚合酶的异常高的焦磷酸解活性表现出这种最后一个方面，只有在模板中存在非天然碱基时才会观察到这种活性。这些结果强调了在引物延伸和焦磷酸解过程中氢键相互作用的重要性。
