Snapshots of a modified nucleotide moving through the confines of a DNA polymerase.
Snapshots of a modified nucleotide moving through the confines of a DNA polymerase.
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DOI：
10.1073/pnas.1811518115
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发表时间：
2018-10-02
影响因子：
11.1
通讯作者：
Marx A
中科院分区：
文献类型：
--
作者：
Kropp HM;Dürr SL;Peter C;Diederichs K;Marx A
尽管DNA聚合酶被进化为处理四种典型的核苷酸，但已知DNA聚合酶可以掺入修饰的核苷酸并从修饰的核苷酸延伸，这是许多核心生物技术应用的关键。掺入后延伸的结构基础仍然难以捉摸。我们成功地将KlenTaq DNA聚合酶结晶成六个复合物，提供了从3′末端上游“移动”到引物链中第六个核苷酸的修饰的高分辨率快照。将这些数据与量子力学/分子力学计算和生物化学研究相结合，阐明了酶和改性底物如何相互调节它们的构象而不影响酶的活性。这突出了该系统作为DNA聚合酶的广泛底物性质的起源的意外可塑性，并指导改进系统的设计。DNA聚合酶已经进化到可以精确地处理这四种典型的核苷酸。尽管如此，这些酶也被称为处理修饰的核苷酸，这是许多核心生物技术应用的关键。修饰的核苷酸的加工包括修饰的核苷酸的掺入和掺入后延伸以继续合成新生DNA链。掺入后延伸的结构基础目前尚不清楚。我们解决了这个问题，并成功地结晶KlenTaq DNA聚合酶在六个封闭的三元复合物含有酶，修饰的DNA底物，和传入的核苷酸。每个结构都显示了修饰引物延伸的高分辨率快照，其中修饰从3′-引物末端上游“移动”到引物链中的第六个核苷酸。将这些数据与量子力学/分子力学计算和生物化学研究相结合，阐明了酶和改性底物如何相互调节它们的构象，而不会显着影响酶的活性。这项研究突出了系统的可塑性作为起源的广泛的底物性质的DNA聚合酶，并有利于设计改进的系统。
Despite being evolved to process the four canonical nucleotides, DNA polymerases are known to incorporate and extend from modified nucleotides, which is the key to numerous core biotechnology applications. The structural basis for postincorporation elongation remained elusive. We successfully crystallized KlenTaq DNA polymerase in six complexes, providing high-resolution snapshots of the modification “moving” from the 3′ terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity. This highlights the unexpected plasticity of the system as origin of the broad substrate properties of the DNA polymerase and guide for the design of improved systems. DNA polymerases have evolved to process the four canonical nucleotides accurately. Nevertheless, these enzymes are also known to process modified nucleotides, which is the key to numerous core biotechnology applications. Processing of modified nucleotides includes incorporation of the modified nucleotide and postincorporation elongation to proceed with the synthesis of the nascent DNA strand. The structural basis for postincorporation elongation is currently unknown. We addressed this issue and successfully crystallized KlenTaq DNA polymerase in six closed ternary complexes containing the enzyme, the modified DNA substrate, and the incoming nucleotide. Each structure shows a high-resolution snapshot of the elongation of a modified primer, where the modification “moves” from the 3′-primer terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity significantly. The study highlights the plasticity of the system as origin of the broad substrate properties of DNA polymerases and facilitates the design of improved systems.
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