Abstract: 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 oriT 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 Bartonella henselae 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
oriT 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
IMPORTANCE 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 in vivo 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.
Otras publicaciones de la misma revista o congreso con autores/as de la Universidad de Cantabria
Fuente: Applied and Environmental Microbiology. June 2017, Volume 83 Issue 12: e00207-17
Editorial: American Society for Microbiology
Fecha de publicación: 01/06/2017
Nº de páginas: 16
Tipo de publicación: Artículo de Revista