Departamento de Biología Molecular, Universidad de Cantabria (UC), Santander, Spain.
Appl Environ Microbiol. 2011 Jan;77(1):201-10. doi: 10.1128/AEM.01371-10. Epub 2010 Oct 29.
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.
TrwC 是一种参与质粒 R388 接合转移的细菌蛋白。它与 DNA 链一起被转移到受体细菌细胞中，在那里它可以将接合转移的 DNA 链整合到受体细胞中存在的靶序列中。考虑到细菌接合可以在细菌和真核细胞之间发生，这种蛋白质作为一种位点特异性整合酶具有巨大的生物技术潜力。我们已经在人类基因组中搜索了可能的 TrwC 靶序列。重组实验表明，TrwC 能够有效地催化其天然靶序列与离散数量的序列之间的重组，这些序列位于人类基因组的非编码位点，类似于该靶序列。我们通过免疫荧光和间接基于酵母的测定法来检测核输入和输出信号，确定了 TrwC 和衍生物在人类细胞中的细胞定位。结果表明，TrwC 的重组酶结构域（N600）具有核定位，但全长 TrwC 位于细胞质中，显然是由于其 C 末端结构域中存在核输出信号。在 TrwC 解旋酶结构域的存在下，TrwC 的重组酶结构域可以通过接合被转运到受体细胞中，但效率非常低。我们突变了 trwC 基因，并选择了具有核定位的突变体。我们获得了一个具有 A904T 点突变和 C 末端额外肽的突变体，它在接合和重组中保持其功能。这种 TrwC 突变体可用于未来在哺乳动物细胞中进行 TrwC 介导的位点特异性整合测定。
