金属离子调控Y家族聚合酶Dpo4合成能力和保真性的机制

在利用DNA作为存储数据介质进行数据写入和读取过程中，易错DNA聚合酶是至关重要的工具酶，其合成能力和保真性易受环境因素所调控。然而，环境因素如何影响合成能力和保真性的调控机制尚缺乏全局性和机理性的认识。本项目以来源于古细菌硫化叶菌(sulfolobus solfataricus)的DNA聚合酶 Dpo4为研究模型，采用计算机模拟和定点突变相结合的方法获得与DNA模板具有不同亲和力/结合自由能的Dpo4突变体，检测这些Dpo4突变体在不同金属离子种类/浓度下的合成能力、保真性和结构特征，从环境因素(离子种类/浓度)-结构特征-合成能力/保真性的关联机制角度阐明金属离子调控DNA聚合酶功能的机制,并在此基础上提出一种筛选合成能力高且保真性随环境离子水平变化的高通量定向进化方法。.研究结果有助于增加对Y家族DNA聚合酶结构与功能的认识，并为同类DNA聚合酶的分子改造提供新方法。

A new idea recently was proposed that DNA could be used as a data storage device, and the error-prone DNA polymerase plays the crucial role in the signal input-output process. In a simple encoding scheme, an environmental signal of interest is coupled to the nucleotide misincorporation rate and the processivity of error-prone DNA polymerase. However, how the environmental signal affecting on the function of the DNA polymerase remains unknown. In this project, DNA polymerase IV(Dpo4) originated from Sulfolobus solfataricus was used as the research model, a library of Dpo4 mutants with different affinity and free energy to DNA template were calculated by computer simulation methods and produced by site-directed mutagenesis. The processivity, fidelity and structural characteristics of these Dpo4 mutants were investigated under different ionic strength conditions. Subsequently, the correlation mechanism of environmental factors- structures-processivity-fidelity was elucidated, and a high-throughput directed evolution method was developed for screening high processivity as well as fidelity changing in ambient ion levels.The research will not only increase the understanding of the structure-function of Y-family DNA polymerases, but also provide a new method for molecular modification of similar DNA polymerase.