Chen Yiqing, Zhang Jing, Liu Hehua, Gao Yanqing, Li Xuhang, Zheng Lina, Cui Ruixue, Yao Qingqing, Rong Liang, Li Jixi, Huang Zhen, Ma Jinbiao, Gan Jianhua
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai, China.
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China.
PLoS Biol. 2017 Feb 28;15(2):e1002599. doi: 10.1371/journal.pbio.1002599. eCollection 2017 Feb.
African swine fever virus (ASFV) can cause highly lethal disease in pigs and is becoming a global threat. ASFV DNA Polymerase X (AsfvPolX) is the most distinctive DNA polymerase identified to date; it lacks two DNA-binding domains (the thumb domain and 8-KD domain) conserved in the homologous proteins. AsfvPolX catalyzes the gap-filling reaction during the DNA repair process of the ASFV virus genome; it is highly error prone and plays an important role during the strategic mutagenesis of the viral genome. The structural basis underlying the natural substrate binding and the most frequent dG:dGTP misincorporation of AsfvPolX remain poorly understood. Here, we report eight AsfvPolX complex structures; our structures demonstrate that AsfvPolX has one unique 5'-phosphate (5'-P) binding pocket, which can favor the productive catalytic complex assembly and enhance the dGTP misincorporation efficiency. In combination with mutagenesis and in vitro catalytic assays, our study also reveals the functional roles of the platform His115-Arg127 and the hydrophobic residues Val120 and Leu123 in dG:dGTP misincorporation and can provide information for rational drug design to help combat ASFV in the future.
非洲猪瘟病毒（ASFV）可导致猪的高致死性疾病，正成为全球威胁。ASFV DNA聚合酶X（AsfvPolX）是迄今为止鉴定出的最独特的DNA聚合酶；它缺乏同源蛋白中保守的两个DNA结合结构域（拇指结构域和8-KD结构域）。AsfvPolX在ASFV病毒基因组的DNA修复过程中催化缺口填充反应；它极易出错，在病毒基因组的策略性诱变过程中发挥重要作用。AsfvPolX天然底物结合以及最常见的dG:dGTP错掺入的结构基础仍知之甚少。在此，我们报告了八个AsfvPolX复合物结构；我们的结构表明，AsfvPolX有一个独特的5'-磷酸（5'-P）结合口袋，其有利于高效催化复合物组装并提高dGTP错掺入效率。结合诱变和体外催化试验，我们的研究还揭示了平台His115-Arg127以及疏水残基Val120和Leu123在dG:dGTP错掺入中的功能作用，并可为未来合理设计药物以对抗ASFV提供信息。
