Nuclear targeting of a bacterial integrase that mediates site-specific recombination between bacterial and human target sequences

Abstract

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.

FIG. 1.

Functional dissection of TrwC and its target oriT sequence. (a) Map of the TrwC protein showing its functional domains. Tra, transfer (functionality in conjugation); Rel, in vitro relaxase activity; Rec, in vivo site-specific recombinase activity; Hel, DNA helicase activity; Dim, dimerization ability. The data were drawn from references and . (b) DNA sequence of the central R388 oriT region (coordinates 201 to 173 from reference 27). The inverted repeat IR₂ is indicated with arrows. Horizontal bars show the minimal sequence requirements for different TrwC activities. bind, in vitro TrwC binding on oligonucleotides; nic, in vitro TrwC nicking and strand-transfer activity on oligonucleotides; recombination, in vivo TrwC-mediated site-specific recombination; transfer, conjugal mobilization (data were drawn from references and 33).

FIG. 2.

Putative TrwC targets in the human genome. (a) DNA sequences resembling the R388 nic site. The minimal region required for TrwC activity is highlighted in boldface. The nic site is indicated by a slash. Arrows show the inverted repeat recognized by TrwC. Nucleotides identical to the R388 sequence are shown in capital letters. The minimal target corresponds to the 173-190 oriT sequence present in plasmid pCIG1073. The mutation altering the R388 nic site in pCIG1110 is shown below the wild-type R388 sequence. Below, the seven human sequences, including the R388 14+2 sequence with a single mismach, are shown, together with shorter sequences mentioned in the text. Sequences are named indicating the human (Hu) chromosome number and the homologous nucleotides 5′+3′ with respect to the nic site; the mismatch position is indicated in parentheses. (b) TrwC binding assays on the (25+8) oligonucleotides containing the sequences shown in panel a. The amount of TrwC-N293 protein in each assay is indicated at the bottom (in nanograms). Arrows point to free (gray arrow) and retarded, TrwC-bound oligonucleotide (black arrow). (c) TrwC strand-transfer reactions on oligonucleotides, including the indicated R388 and human sequences. Arrows point to the cut product (gray arrow) or strand transfer product (black arrow), which is quantitated at the bottom of the gel (%ST). The assays for panels b and c were performed as described in Materials and Methods.

FIG. 3.

Typical colonies obtained with recombination substrates containing the indicated sequences (as shown in Fig. 2a) cloned at oriT1. oriT2 remains invariable and contains the full-length original R388 oriT. Recombination assays were performed in DH5α strain in the presence of a plasmid coding for both TrwA and TrwC.

FIG. 4.

Localization of TrwC and derivatives in human cells. Immunofluorescence images of 293T cells transduced with plasmids coding for TrwC or the indicated TrwC segments. Images are shown at ×40 magnification.

FIG. 5.

Yeast nuclear import/export assays. (a) Scheme of the yeast nuclear import assay. The test protein (green circle) is fused to transcriptional regulators, which turn on reporter genes if driven into the nucleus, thus allowing the expression of HIS3 and lacZ. Selection is illustrated in the right panels. HIS3 expression allows growth in histidine-deficient medium (−His), and lacZ is monitored by β-galactosidase activity in cells transferred to nitrocellulose filters with X-Gal (NC+X-Gal). (b) Results of the nuclear import (left two panels) and export (right two panels) assay with TrwC and derivatives. The numbers on the plates in panels a and b represent the following: 1, pNIA:VirD2; 2, pNIA:E2; 3, pNIA:N293; 4, pNIA:C774; 5, pNIA:TrwC; 6, pNIA:N600; 7, pNEA:VirE2; 8, pNEA:TrwC; and 9, pNEA:C774.

FIG. 6.

TrwC mutants for nuclear targeting. (a) Recombination assays. DH5α cells containing the recombination substrate pCIG1030 (including trwA) and the plasmid coding for the indicated TrwC fusion derivative were plated on X-Gal containing selective media. TrwC* refers to the TrwC mutant coded by plasmid pLA44. (b) Yeast nuclear import assays (as in Fig. 5). C+ and C− refer to pNIA:VirD2 and pNIA:VirE2, respectively. The numbers on the plates represent the following: 1, pNIA:TrwC; 2, pNIA:TrwC* (TrwC mutant coded by pLA44); 3, pNIA:TrwC-NLS; and 4, pNIA:NLS(x2)-TrwC.