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Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering

Transposable elements are efficient DNA carriers and thus important tools for transgenesis and insertional mutagenesis. However, their poor target sequence specificity constitutes an important limitation for site-directed applications. The insertion sequence IS608 from Helicobacter pylori recognizes...

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Autores principales: Morero, Natalia Rosalía, Zuliani, Cecilia, Kumar, Banushree, Bebel, Aleksandra, Okamoto, Sachi, Guynet, Catherine, Hickman, Alison Burgess, Chandler, Michael, Dyda, Fred, Barabas, Orsolya
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934647/
https://www.ncbi.nlm.nih.gov/pubmed/29635476
http://dx.doi.org/10.1093/nar/gky235
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author Morero, Natalia Rosalía
Zuliani, Cecilia
Kumar, Banushree
Bebel, Aleksandra
Okamoto, Sachi
Guynet, Catherine
Hickman, Alison Burgess
Chandler, Michael
Dyda, Fred
Barabas, Orsolya
author_facet Morero, Natalia Rosalía
Zuliani, Cecilia
Kumar, Banushree
Bebel, Aleksandra
Okamoto, Sachi
Guynet, Catherine
Hickman, Alison Burgess
Chandler, Michael
Dyda, Fred
Barabas, Orsolya
author_sort Morero, Natalia Rosalía
collection PubMed
description Transposable elements are efficient DNA carriers and thus important tools for transgenesis and insertional mutagenesis. However, their poor target sequence specificity constitutes an important limitation for site-directed applications. The insertion sequence IS608 from Helicobacter pylori recognizes a specific tetranucleotide sequence by base pairing, and its target choice can be re-programmed by changes in the transposon DNA. Here, we present the crystal structure of the IS608 target capture complex in an active conformation, providing a complete picture of the molecular interactions between transposon and target DNA prior to integration. Based on this, we engineered IS608 variants to direct their integration specifically to various 12/17-nt long target sites by extending the base pair interaction network between the transposon and the target DNA. We demonstrate in vitro that the engineered transposons efficiently select their intended target sites. Our data further elucidate how the distinct secondary structure of the single-stranded transposon intermediate prevents extended target specificity in the wild-type transposon, allowing it to move between diverse genomic sites. Our strategy enables efficient targeting of unique DNA sequences with high specificity in an easily programmable manner, opening possibilities for the use of the IS608 system for site-specific gene insertions.
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spelling pubmed-59346472018-05-09 Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering Morero, Natalia Rosalía Zuliani, Cecilia Kumar, Banushree Bebel, Aleksandra Okamoto, Sachi Guynet, Catherine Hickman, Alison Burgess Chandler, Michael Dyda, Fred Barabas, Orsolya Nucleic Acids Res Nucleic Acid Enzymes Transposable elements are efficient DNA carriers and thus important tools for transgenesis and insertional mutagenesis. However, their poor target sequence specificity constitutes an important limitation for site-directed applications. The insertion sequence IS608 from Helicobacter pylori recognizes a specific tetranucleotide sequence by base pairing, and its target choice can be re-programmed by changes in the transposon DNA. Here, we present the crystal structure of the IS608 target capture complex in an active conformation, providing a complete picture of the molecular interactions between transposon and target DNA prior to integration. Based on this, we engineered IS608 variants to direct their integration specifically to various 12/17-nt long target sites by extending the base pair interaction network between the transposon and the target DNA. We demonstrate in vitro that the engineered transposons efficiently select their intended target sites. Our data further elucidate how the distinct secondary structure of the single-stranded transposon intermediate prevents extended target specificity in the wild-type transposon, allowing it to move between diverse genomic sites. Our strategy enables efficient targeting of unique DNA sequences with high specificity in an easily programmable manner, opening possibilities for the use of the IS608 system for site-specific gene insertions. Oxford University Press 2018-05-04 2018-04-09 /pmc/articles/PMC5934647/ /pubmed/29635476 http://dx.doi.org/10.1093/nar/gky235 Text en © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Nucleic Acid Enzymes
Morero, Natalia Rosalía
Zuliani, Cecilia
Kumar, Banushree
Bebel, Aleksandra
Okamoto, Sachi
Guynet, Catherine
Hickman, Alison Burgess
Chandler, Michael
Dyda, Fred
Barabas, Orsolya
Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering
title Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering
title_full Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering
title_fullStr Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering
title_full_unstemmed Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering
title_short Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering
title_sort targeting is608 transposon integration to highly specific sequences by structure-based transposon engineering
topic Nucleic Acid Enzymes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934647/
https://www.ncbi.nlm.nih.gov/pubmed/29635476
http://dx.doi.org/10.1093/nar/gky235
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