TrwC-Mediated Site-Specific Recombination Is Controlled by Host Factors Altering Local DNA Topology

César, Carolina Elvira; Llosa, Matxalen

doi:10.1128/jb.01152-07

Cited by 10 publications

(9 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…With respect to TrwC-mediated oriT -specific recombination on scDNA substrates, TrwA increases drastically the efficiency of the reaction in the absence of conjugation, and it also enhances TrwC-mediated site-specific recombination in the recipient cell by almost six times [15]. IHF was shown to inhibit recombination only in the absence of TrwA and on DNA substrates lacking one of the two TrwA and IHF binding sites [16]; in the presence of TrwA, however, the presence or absence of IHF was irrelevant.…”

Section: Resultsmentioning

confidence: 99%

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

et al. 2012

Self Cite

View full text Add to dashboard Cite

BackgroundBacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering.Methodology/Principal FindingsWe have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome.Conclusions/SignificanceThe results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.

show abstract

Section: Resultsmentioning

confidence: 99%

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

et al. 2012

Self Cite

View full text Add to dashboard Cite

show abstract

“…We transfected both EA.hy926 and HeLa cells with plasmids pMTX708 and pMTX709 (Table 1) carrying a hygromycin resistance gene and the R388 oriT in both orientations. Both plasmids were used to avoid any bias due to eukaryotic promoters present in the vector, since transcription through oriT has been shown to affect TrwC-mediated recombination (11), and so it might affect integration. Around 100 hygromycin-resistant colonies were obtained in transfections of HeLa cells, while no transfectants appeared for EA.hy926 in spite of several attempts with up to 5 g of plasmid DNA.…”

Section: Construction Of Mobilizable Plasmids and Target Cell Linesmentioning

confidence: 99%

“…The reaction takes place in the absence of conjugation and thus in the absence of single-stranded intermediates, and it is favored by the accessory protein TrwA. In contrast, IHF was found to exert a negative regulatory role in TrwC-mediated recombination (11). It was proposed that recombination takes place thanks to the single-stranded endonuclease activity of TrwC coupled to the replication machinery of the host cell (10).…”

mentioning

confidence: 99%

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

González-Prieto

Rinaldi

Dehio

et al. 2017

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used to transfer the DNA of interest into the appropriate cell and promote its integration. If used in combination with a site-specific nuclease, it could lead to site-specific integration of the incoming DNA by homologous recombination.

show abstract

“…In most documented cases, auxiliary proteins of both host and plasmid origins assist the relaxase, allowing the formation of a DNA-protein complex known as the relaxosome (14,42). Plasmid oriTs generally contain features, such as inverted repeats and A tracts, that are located near the plasmid strand cleavage site and form hairpins when in single-stranded form (19).…”

mentioning

confidence: 99%

Identification of the Origin of Transfer (oriT) and a New Gene Required for Mobilization of the SXT/R391 Family of Integrating Conjugative Elements

et al. 2008

View full text Add to dashboard Cite

Integrating conjugative elements (ICEs) are self-transmissible, mobile elements that are widespread among bacteria. Following their excision from the chromosome, ICEs transfer by conjugation, a process initiated by a single-stranded DNA break at a specific locus called the origin of transfer (oriT). The SXT/R391 family of ICEs includes SXT MO10 , R391, and more than 25 related ICEs found in gammaproteobacteria. A previous study mapped the oriT locus of SXT MO10 to a 550-bp intergenic region between traD and s043. We suspected that this was not the correct oriT locus, because the identical traD-s043 region in R391 and other SXT/R391 family ICEs was annotated as a gene of an unknown function. Here, we investigated the location and structure of the oriT locus in the ICEs of the SXT/R391 family and demonstrated that oriT SXT corresponds to a 299-bp sequence that contains multiple imperfect direct and inverted repeats and is located in the intergenic region between s003 and rumB. The oriT SXT locus is well conserved among SXT/R391 ICEs, like R391, R997, and pMERPH, and cross-recognition of oriT SXT and oriT R391 by R391 and SXT MO10 was demonstrated. Furthermore, we identified a previously unannotated gene, mobI, located immediately downstream from oriT SXT , which proved to be essential for SXT MO10 transfer and SXT MO10 -mediated chromosomal DNA mobilization. Deletion of mobI did not impair the SXT MO10 -dependent transfer of the mobilizable plasmid CloDF13, suggesting that mobI has no role in the assembly of the SXT MO10 mating pair apparatus. Instead, mobI appears to be involved in the recognition of oriT SXT .Integrating conjugative elements (ICEs) are a large family of self-transmissible mobile genetic elements that are widespread among bacteria (8,12). These elements confer a range of properties upon the host bacteria, including resistance to antibiotics and heavy metals, virulence, symbiosis establishment, and alternative metabolic pathways. ICEs are made of a core set of genes ensuring their maintenance, mobility, and regulation (8,12,44,50).Following their excision from the chromosome of the host cell as a circular intermediate, ICEs transfer by conjugation, using a mechanism similar to that of conjugative plasmids (8,15,19,23,32,39). Integration into and excision from the chromosome occur by recombination mediated by an ICEencoded site-specific recombinase called integrase (Int) (10). Several ICEs are able to mobilize nonconjugative plasmids in cis and in trans as well as chromosomal DNA in an Hfr-like manner (27).Conjugative DNA transfer takes place in two key steps: (i) biochemical processing of the DNA molecule for transfer and (ii) assembly of a mating apparatus bridging the donor and recipient cells to allow DNA transfer. Typically conjugative DNA transfer is initiated at a specific cis-acting site called the origin of transfer (oriT), required for efficient translocation of the DNA to the recipient cell (23,39). A DNA relaxase encoded by the conjugative element recognizes the oriT locus and cleaves o...

show abstract

TrwC-Mediated Site-Specific Recombination Is Controlled by Host Factors Altering Local DNA Topology

Cited by 10 publications

References 31 publications

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

Identification of the Origin of Transfer (oriT) and a New Gene Required for Mobilization of the SXT/R391 Family of Integrating Conjugative Elements

Contact Info

Product

Resources

About