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

AuthorsAgúndez, Leticia ; Machón, Cristina ; Elvira César, Carolina ; Rosa-Garrido, Manuel ; Delgado, M. Dolores ; Llosa, Matxalen
Issue Date2011
PublisherAmerican Society for Microbiology
CitationApplied and Environmental Microbiology 77(1): 201-210 (2010)
AbstractTrwC 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.
DescriptionEl pdf del artículo es la versión pre-print.-- Trabajo presentado a la: "IV International Conference on Environmental, Industrial and Applied Microbiology (BioMicroWorld2011)" celebrada del 14 al 16 de septiembre de 2011 en Torremolinos (España).
Publisher version (URL)http://dx.doi.org/10.1128/​AEM.01371-10
Appears in Collections:(IBBTEC) Artículos
(CNB) Artículos
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