Cargando…

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

BACKGROUND: Bacterial 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...

Descripción completa

Detalles Bibliográficos
Autores principales: Agúndez, Leticia, González-Prieto, Coral, Machón, Cristina, Llosa, Matxalen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264647/
https://www.ncbi.nlm.nih.gov/pubmed/22292089
http://dx.doi.org/10.1371/journal.pone.0031047
_version_ 1782222006592208896
author Agúndez, Leticia
González-Prieto, Coral
Machón, Cristina
Llosa, Matxalen
author_facet Agúndez, Leticia
González-Prieto, Coral
Machón, Cristina
Llosa, Matxalen
author_sort Agúndez, Leticia
collection PubMed
description BACKGROUND: Bacterial 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 FINDINGS: We 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/SIGNIFICANCE: The 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.
format Online
Article
Text
id pubmed-3264647
institution National Center for Biotechnology Information
language English
publishDate 2012
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-32646472012-01-30 Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase Agúndez, Leticia González-Prieto, Coral Machón, Cristina Llosa, Matxalen PLoS One Research Article BACKGROUND: Bacterial 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 FINDINGS: We 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/SIGNIFICANCE: The 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. Public Library of Science 2012-01-23 /pmc/articles/PMC3264647/ /pubmed/22292089 http://dx.doi.org/10.1371/journal.pone.0031047 Text en Agúndez et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Agúndez, Leticia
González-Prieto, Coral
Machón, Cristina
Llosa, Matxalen
Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase
title Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase
title_full Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase
title_fullStr Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase
title_full_unstemmed Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase
title_short Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase
title_sort site-specific integration of foreign dna into minimal bacterial and human target sequences mediated by a conjugative relaxase
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264647/
https://www.ncbi.nlm.nih.gov/pubmed/22292089
http://dx.doi.org/10.1371/journal.pone.0031047
work_keys_str_mv AT agundezleticia sitespecificintegrationofforeigndnaintominimalbacterialandhumantargetsequencesmediatedbyaconjugativerelaxase
AT gonzalezprietocoral sitespecificintegrationofforeigndnaintominimalbacterialandhumantargetsequencesmediatedbyaconjugativerelaxase
AT machoncristina sitespecificintegrationofforeigndnaintominimalbacterialandhumantargetsequencesmediatedbyaconjugativerelaxase
AT llosamatxalen sitespecificintegrationofforeigndnaintominimalbacterialandhumantargetsequencesmediatedbyaconjugativerelaxase