Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering
| Autor: | Banushree Kumar, Alison B. Hickman, Michael Chandler, Catherine Guynet, Natalia Rosalía Morero, Sachi Okamoto, Orsolya Barabas, Cecilia Zuliani, Aleksandra Bebel, Fred Dyda |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
DNA
Bacterial Models Molecular 0301 basic medicine Transposable element Base pair Transposases Computational biology Biology DNA sequencing Insertional mutagenesis 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Genetics Insertion sequence Base Pairing Transposase Base Sequence Helicobacter pylori Nucleic Acid Enzymes Transposon integration 030104 developmental biology chemistry DNA Transposable Elements Nucleic Acid Conformation Genetic Engineering 030217 neurology & neurosurgery DNA |
| Zdroj: | Nucleic Acids Research 'Nucleic Acids Research ', vol: 46, pages: 4152-4163 (2018) |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gky235 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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