TcBuster is a hAT-family DNA transposon from the red flour beetle, Tribolium castaneum. The TcBuster transposase is of interest for genome engineering as it is highly active in insect and mammalian cells. To test the predicted catalytic triad of TcBuster, each residue of the catalytic triad of a haemagglutinin-tagged TcBuster transposase was individually mutated to a structurally conserved amino acid. Using a drug-resistant colony assay for transposon integration, we found that the D223N, D289N, and E589Q mutants of TcBuster transposase were inactive in human cells. We used a modified chromatin immunoprecipitation assay to determine that each mutant maintained binding to TcBuster transposon inverted repeat elements. Although the catalytic mutants retained their transposon binding properties, mutants displayed altered expression and localization in human cells. None of the catalytic mutants formed characteristic TcBuster transposase rodlet structures, and the D223N and D289N mutants were not able to be detected by immunofluorescence microscopy. Immunoblot analysis demonstrated that the E589Q mutant is less abundant than wild-type TcBuster transposase. Cells transfected with either TcBuster or TcBuster-E589Q transposase were imaged by structured illumination microscopy to quantify differences in the length of the transposase rodlets. The average length of the TcBuster transposase rodlets (N = 39) was 3.284 μm while the E589Q rodlets (N = 33) averaged 1.157 μm (p < 0.0001; t-test). The catalytic triad mutations decreased overall protein levels and disrupted transposase rodlet formation while nuclear localization and DNA binding to the inverted repeat elements were maintained. Our results may have broader implications for the overproduction inhibition phenomenon observed for DNA transposons.

TcBuster是来自红色面粉甲虫Tribolium castaneum的hAT家族 DNA 转座子。TcBuster转座酶对基因组工程很感兴趣，因为它在昆虫和哺乳动物细胞中具有高度活性。为了测试预测的TcBuster催化三联体，血凝素标记的TcBuster转座酶的催化三联体的每个残基分别突变为结构上保守的氨基酸。使用耐药菌落检测转座子整合，我们发现 TcBuster 的 D223N、D289N 和 E589Q 突变体转座酶在人体细胞中是无活性的。我们使用改进的染色质免疫沉淀测定来确定每个突变体保持与TcBuster转座子反向重复元件的结合。尽管催化突变体保留了它们的转座子结合特性，但突变体在人类细胞中表现出改变的表达和定位。没有一个催化突变体形成特征性的TcBuster转座酶棒状结构，并且 D223N 和 D289N 突变体无法通过免疫荧光显微镜检测到。免疫印迹分析表明，E589Q 突变体的丰度低于野生型TcBuster转座酶。用TcBuster或TcBuster转染的细胞-E589Q 转座酶通过结构化照明显微镜成像，以量化转座酶小棒长度的差异。TcBuster转座酶小棒 ( N = 39)的平均长度 为 3.284 微米，而 E589Q 小棒 ( N  = 33) 的平均长度为 1.157 微米 ( p  < 0.0001；t检验)。催化三联体突变降低了整体蛋白质水平并破坏了转座酶小柱的形成，而核定位和 DNA 与反向重复元件的结合得以维持。我们的结果可能对观察到的 DNA 转座子的过度生产抑制现象具有更广泛的意义。