Department of Chemistry and Biochemistry, Ohio State Biochemistry Program , The Ohio State University , Columbus , Ohio 43210 , United States.
Chem Rev. 2018 Jun 27;118(12):6000-6025. doi: 10.1021/acs.chemrev.7b00685. Epub 2018 Jun 4.
Faithful transmission and maintenance of genetic material is primarily fulfilled by DNA polymerases. During DNA replication, these enzymes catalyze incorporation of deoxynucleotides into a DNA primer strand based on Watson-Crick complementarity to the DNA template strand. Through the years, research on DNA polymerases from every family and reverse transcriptases has revealed structural and functional similarities, including a conserved domain architecture and purported two-metal-ion mechanism for nucleotidyltransfer. However, it is equally clear that DNA polymerases possess distinct differences that often prescribe a particular cellular role. Indeed, a unified kinetic mechanism to explain all aspects of DNA polymerase catalysis, including DNA binding, nucleotide binding and incorporation, and metal-ion-assisted nucleotidyltransfer (i.e., chemistry), has been difficult to define. In particular, the contributions of enzyme conformational dynamics to several mechanistic steps and their implications for replication fidelity are complex. Moreover, recent time-resolved X-ray crystallographic studies of DNA polymerases have uncovered a third divalent metal ion present during DNA synthesis, the function of which is currently unclear and debated within the field. In this review, we survey past and current literature describing the structures and kinetic mechanisms of DNA polymerases from each family to explore every major mechanistic step while emphasizing the impact of enzyme conformational dynamics on DNA synthesis and replication fidelity. This also includes brief insight into the structural and kinetic techniques utilized to study DNA polymerases and RTs. Furthermore, we present the evidence for the two-metal-ion mechanism for DNA polymerase catalysis prior to interpreting the recent structural findings describing a third divalent metal ion. We conclude by discussing the diversity of DNA polymerase mechanisms and suggest future characterization of the third divalent metal ion to dissect its role in DNA polymerase catalysis.
遗传物质的忠实传递和维持主要由 DNA 聚合酶来完成。在 DNA 复制过程中，这些酶根据 DNA 模板链与 DNA 引物链之间的 Watson-Crick 互补性，催化脱氧核苷酸掺入到 DNA 引物链中。多年来，对来自每个家族的 DNA 聚合酶和逆转录酶的研究揭示了结构和功能上的相似性，包括保守的结构域架构和假定的双金属离子机制用于核苷酸转移。然而，同样清楚的是，DNA 聚合酶具有明显的差异，这些差异通常规定了特定的细胞作用。事实上，很难定义一个统一的动力学机制来解释 DNA 聚合酶催化的所有方面，包括 DNA 结合、核苷酸结合和掺入以及金属离子辅助的核苷酸转移（即化学）。特别是，酶构象动力学对几个机制步骤的贡献及其对复制保真度的影响是复杂的。此外，最近对 DNA 聚合酶的时间分辨 X 射线晶体学研究揭示了在 DNA 合成过程中存在第三种二价金属离子，其功能目前在该领域尚不清楚且存在争议。在这篇综述中，我们调查了过去和当前的文献，描述了每个家族的 DNA 聚合酶的结构和动力学机制，以探索每个主要的机制步骤，同时强调酶构象动力学对 DNA 合成和复制保真度的影响。这还包括对用于研究 DNA 聚合酶和 RT 的结构和动力学技术的简要了解。此外，我们提出了 DNA 聚合酶催化的双金属离子机制的证据，然后再解释描述第三种二价金属离子的最近结构发现。最后，我们讨论了 DNA 聚合酶机制的多样性，并建议对第三种二价金属离子进行未来的特征描述，以剖析其在 DNA 聚合酶催化中的作用。
