Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase.
Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase.
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DOI：
10.1016/j.jbc.2022.101627
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发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Johnson KA
中科院分区：
文献类型：
--
作者：
Dangerfield TL;Kirmizialtin S;Johnson KA
高保真 DNA 聚合酶对基因组 DNA 的忠实复制对于大多数生物体的生存至关重要。虽然高保真度 DNA 聚合酶有利于规范碱基对，而不是错配，其保真度可通过 3'–5' 校对核酸外切酶进一步增强几个数量级，该酶选择性地去除引物链中的错配碱基。尽管校对对于维持基因组稳定性很重要，但与聚合酶活性位点采用的保真度机制相比，对它的研究仍然少得多。在这里，我们通过研究各种 DNA 底物的校对动力学来表征高保真 DNA 聚合酶的校对核酸外切酶的底物特异性。核酸外切酶对净保真度的贡献是延伸和切除之间的动力学分配的函数。我们证明，虽然对末端不匹配的校对是有效的，但对被一两个正确碱基掩埋的不匹配进行校对甚至更有效。因为聚合酶在错配掺入后以及在错配之上掺入一两个正确碱基后停止，所以核酸外切酶的净贡献是纠正错误的多个机会的函数。我们还使用硫代磷酸酯修饰的 DNA 表征了核酸外切酶的立体特异性，提供了核酸外切酶活性位点中 DNA 引物链的同源模型，并提出了基于 MD 模拟的 DNA 从聚合酶转移到核酸外切酶活性位点的动态结构模型。
Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 105, fidelity is further enhanced several orders of magnitude by a 3′–5′ proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations.
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