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首页> 外文期刊>Applied Microbiology >Nuclear Targeting of a Bacterial Integrase That Mediates Site-Specific Recombination between Bacterial and Human Target Sequences
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Nuclear Targeting of a Bacterial Integrase That Mediates Site-Specific Recombination between Bacterial and Human Target Sequences

机译：介导细菌和人类靶序列之间特定位点重组的细菌整合酶的核靶向。

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TrwC is a bacterial protein involved in conjugative transfer of plasmid R388. It is transferred together with the DNA strand into the recipient bacterial cell, where it can integrate the conjugatively transferred DNA strand into its target sequence present in the recipient cell. Considering that bacterial conjugation can occur between bacteria and eukaryotic cells, this protein has great biotechnological potential as a site-specific integrase. We have searched for possible TrwC target sequences in the human genome. Recombination assays showed that TrwC efficiently catalyzes recombination between its natural target sequence and a discrete number of sequences, located in noncoding sites of the human genome, which resemble this target. We have determined the cellular localization of TrwC and derivatives in human cells by immunofluorescence and also by an indirect yeast-based assay to detect both nuclear import and export signals. The results indicate that the recombinase domain of TrwC (N600) has nuclear localization, but full-length TrwC locates in the cytoplasm, apparently due to the presence of a nuclear export signal in its C-terminal domain. The recombinase domain of TrwC can be transported to recipient cells by conjugation in the presence of the helicase domain of TrwC, but with very low efficiency. We mutagenized the trwC gene and selected for mutants with nuclear localization. We obtained one such mutant with a point A904T mutation and an extra peptide at its C terminus, which maintained its functionality in conjugation and recombination. This TrwC mutant could be useful for future TrwC-mediated site-specific integration assays in mammalian cells.
机译：TrwC是一种细菌蛋白，参与质粒R388的共轭转移。它与DNA链一起转移到受体细菌细胞中，在这里它可以将结合转移的DNA链整合到受体细胞中存在的靶序列中。考虑到细菌与真核细胞之间可能发生细菌结合，该蛋白作为位点特异性整合酶具有巨大的生物技术潜力。我们已经搜索了人类基因组中可能的TrwC靶序列。重组分析表明，TrwC有效催化其天然靶序列与位于人类基因组非编码位点（类似于该靶点）中的离散序列之间的重组。我们已经通过免疫荧光法以及通过基于酵母的间接检测来检测核输入和输出信号，确定了TrwC及其衍生物在人细胞中的细胞定位。结果表明，TrwC（N600）的重组酶结构域具有核定位，但是全长TrwC位于细胞质中，这显然是由于其C端结构域中存在核输出信号。 TrwC的重组酶结构域可以在TrwC的解旋酶结构域存在的情况下通过缀合运输到受体细胞，但是效率非常低。我们诱变了trwC基因，并选择了具有核定位的突变体。我们获得了一个这样的突变体，该突变体具有A904T点突变，并在其C末端带有一个额外的肽，该肽在缀合和重组中保持了其功能。该TrwC突变体可用于未来TrwC介导的哺乳动物细胞中的位点特异性整合测定。

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