金属离子调控Y家族聚合酶Dpo4合成能力和保真性的机制

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.

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。

Y家族DNA聚合酶是一类能够以损伤DNA为模板，跨越损伤位点进行DNA复制的聚合酶，它能够跨越不同类型的DNA损伤，帮助细胞维持基因组稳定性，使细胞能够在恶劣条件下生长。该项目在对Y家族DNA聚合酶Dpo4进行定向改造的基础上，获得合成能力提高的突变体并探究Dpo4中不同结构域与Dpo4合成能力的关系，对突变体的保真性、核苷酸掺入效率及跨损伤合成能力展开研究，阐明结构域对Dpo4整体性能的影响，主要研究结果如下：(1)筛选出10个潜在突变体，包括finger区域的F33Y、F37T、I59M、E63K、M76I，thumb区域的A181D、N188S、A220S，LF区域的I248Y、V289I，进一步计算wtDpo4及突变体与DNA的结合能，获得8个结合能增强的突变体进行异源表达和分离纯化；(2)wtDpo4及突变体的合成能力检测结果表明，A181D合成能力最好，相比wtDpo4增加了25%；(3)比较位于thumb、finger、LF区域的突变体A181D、E63K、I248Y与wtDpo4的保真性及核苷酸掺入效率，结果表明A181D的保真性最低，I248Y最高，wtDpo4、A181D、E63K、V289I的核苷酸掺入效率分别为4.18×10-4 s-1、4.74×10-4 s-1、4.37×10-4 s-1和2.09×10-4 s-1μM-1，表明A181D的核苷酸掺入效率最高；(4)比较A181D与wtDpo4跨越损伤碱基8-oxoG的合成能力：在损伤碱基8-oxoG上掺入dCTP时，A181D 和wtDpo4的催化效率分别为4.19×10-2和1.10×10-2 μM-1min-1；在掺入dATP时，两者分别为0.34×10-2 和0.13×10-2 μM-1min-1；跨越损伤碱基对8-oxoG:C时，两者分别为1.69×10-2和1.09×10-2 μM-1 min-1，突变体A181D的跨损伤合成能力相比wtDpo4提高; (5)进一步对Dpo4在真核生物酿酒酵母中的同系物Polη和Rev1在细胞内应答氧胁迫的作用机制进行了研究，发现缺失Polη降低菌株在氧胁迫下生存能力，同时核糖核苷酸还原酶抑制剂Sml1通过36-70位氨基酸与Rev1的BRCT结构域相互作用，抑制Rev1在氧胁迫中的修复功能。