Increased Processivity, Misincorporation, and Nucleotide Incorporation Efficiency in Sulfolobus solfataricus Dpo4 Thumb Domain Mutants
Increased Processivity, Misincorporation, and Nucleotide Incorporation Efficiency in Sulfolobus solfataricus Dpo4 Thumb Domain Mutants
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硫磺硫化叶菌 Dpo4 Thumb 结构域突变体的持续合成能力、错误掺入和核苷酸掺入效率提高
DOI：
10.1128/aem.01013-17
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发表时间：
2017-07
影响因子：
4.4
通讯作者：
Tyo Keith E. J.
中科院分区：
文献类型：
--
作者：
Wang Li;Liang Chenchen;Wu Jing;Liu Liming;Tyo Keith E. J.
摘要本研究旨在提高黄柳草DNA聚合酶Dpo4的活性。利用蛋白质工程和生物信息学技术建立了Dpo4潜在突变位点文库。鉴定并构建了10个潜在突变体。用引物延伸试验评价了Dpo4突变体的加工性。拇指（A181D）和手指（E63K）结构域突变体分别具有20和19个核苷酸（nt）的加工能力。小指结构域突变体I248Y的加工性为17 nt，仅比野生型Dpo4高1 nt。此外，与E63K和I248Y突变体相比，A181D突变体具有较低的保真度和较高的核苷酸整合效率（4.74 × 10−4 s−1 μM−1）。当对A181D突变体进行旁路损伤处理时，与野生型Dpo4相比，A181D突变体结合dCTP和dATP的催化效率（kcat/Km）分别高出3.81倍和2.62倍。与野生型Dpo4相比，当它越过受损的8-oxoG:C和8-oxoG: a碱基对时，其催化效率分别提高了55%和91.5%。蛋白质工程和生物信息学方法可以有效地提高Dpo4的加工能力和翻译合成能力。重要性：保真度较差的DNA聚合酶可用于存储数据和记录响应于细胞内环境的变化。硫酸亚硫酸盐Dpo4就是这样一种酶，尽管它的使用受到其低加工能力的阻碍。在这项工作中，我们使用生物信息学和蛋白质工程方法来产生具有改进加工能力的Dpo4突变体。我们确定Dpo4拇指结构域在控制处理能力方面最相关。
ABSTRACT The present study aimed to increase the processivity of Sulfolobus solfataricus DNA polymerase Dpo4. Protein engineering and bioinformatics were used to compile a library of potential Dpo4 mutation sites. Ten potential mutants were identified and constructed. A primer extension assay was used to evaluate the processivity of Dpo4 mutants. Thumb (A181D) and finger (E63K) domain mutants showed a processivity of 20 and 19 nucleotides (nt), respectively. A little finger domain mutant (I248Y) exhibited a processivity of 17 nt, only 1 nt more than wild-type Dpo4. Furthermore, the A181D mutant showed lower fidelity and higher nucleotide incorporation efficiency (4.74 × 10−4 s−1 μM−1) than E63K and I248Y mutants. When tasked with bypassing damage, the A181D mutant exhibited a 3.81-fold and 2.62-fold higher catalytic efficiency (kcat/Km) at incorporating dCTP and dATP, respectively, than wild-type Dpo4. It also showed a 55% and 91.5% higher catalytic efficiency when moving beyond the damaged 8-oxoG:C and 8-oxoG:A base pairs, respectively, compared to wild-type Dpo4. Protein engineering and bioinformatics methods can effectively increase the processivity and translesion synthesis ability of Dpo4. IMPORTANCE DNA polymerases with poor fidelity can be exploited to store data and record changes in response to the intracellular environment. Sulfolobus solfataricus Dpo4 is such an enzyme, although its use is hindered by its low processivity. In this work, we used a bioinformatics and protein engineering approach to generate Dpo4 mutants with improved processivity. We identified the Dpo4 thumb domain as the most relevant in controlling processivity.
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影响因子：
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