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Retargeting of the IS608 Transposon

Kumar, Banushree (2015) MOBM01 20151
Degree Projects in Molecular Biology
Abstract
DNA transposons are a class of mobile genetic elements that can autonomously move from one genomic location to another. They are powerful drivers of genetic change and have played a significant role in the evolution of many genomes. One such transposable element, IS608 from Helicobacter pylori employs a unique mechanism of transposition as it transposes in a single-stranded DNA form and inserts specifically 3’ of a specific tetranucleotide sequence (Kersulyte et al., 2002; Guynet et al., 2008). Previous structural (Ronning et al., 2005; Barabas et al, 2008) and biochemical studies (Ton-Hoang et al., 2005) of IS608, revealed that the element chooses its integration site specifically via base-pairing between the transposon and the target... (More)
DNA transposons are a class of mobile genetic elements that can autonomously move from one genomic location to another. They are powerful drivers of genetic change and have played a significant role in the evolution of many genomes. One such transposable element, IS608 from Helicobacter pylori employs a unique mechanism of transposition as it transposes in a single-stranded DNA form and inserts specifically 3’ of a specific tetranucleotide sequence (Kersulyte et al., 2002; Guynet et al., 2008). Previous structural (Ronning et al., 2005; Barabas et al, 2008) and biochemical studies (Ton-Hoang et al., 2005) of IS608, revealed that the element chooses its integration site specifically via base-pairing between the transposon and the target DNA. This unique feature allowed re-directing transposon integration to various four-nucleotide sequences by simply modifying the transposon DNA sequence. A key feature of the retargeting strategy was that, unlike the previous attempts to retarget mobile elements, the target specificity of IS608 could be varied straightforwardly by altering the transposon sequence without any need to modify the transposase protein.
In this study, I tested if the target selection mechanism of IS608 could be exploited to direct the transposon specifically to longer DNA sequences, which could potentially be unique in the context of a genome. To this end, I first used an oligonucleotide based retargeting assay in vitro, where both the transposon and target sequences were engineered to allow specific selection of 12-nucleotide target sequences. The results of this assay showed that IS608 can be retargeted in a programmable way to multiple sequences in vitro. Additionally, the activity and selectivity of transposon integration could be further improved by the presence of a specific nucleotide adjacent to the TTAC in the target sequence, which revealed a novel feature of the IS608 transposition mechanism. Currently, we are pursuing crystallization of IS608 transposase-transposon-target DNA complexes that will further elucidate the molecular principles of this finding. Candidate transposon sequence variants were taken forward to an in vivo mating out assay in E. coli. In this ongoing assay, the rates of transposon integration at specific target sequences will be calculated and compared to the integration at non-specific targets, giving an estimate of the retargeting efficiency. In summary, a combination of molecular biology techniques, including structural biology, biochemistry, and microbiology, allowed me to contribute to the body of knowledge regarding IS608’s integration mechanism, which will hopefully in the future allow the development of a new, programmable tool for functional genomics, synthetic biology, and medicinal genetics. (Less)
Popular Abstract
Programmable gene jumping: the IS608 case

DNA transposons are segments of DNA that can jump from one position to another in the genome of a single cell. For this movement to occur, the transposon codes for a protein called the transposase that catalyses the transfer of the DNA from the donor location to a new randomly selected target location. The subject of this study, the IS608 transposon from the gastric pathogen Helicobacter pylroi is, however, unique in this regard. It only deals with a single strand of DNA throughout its pathway and inserts specifically at a ‘TTAC’ nucleotide sequence. In the presented work, we show that the target specificity of IS608 can be further increased by simply modifying parts of the transposon sequence.
... (More)
Programmable gene jumping: the IS608 case

DNA transposons are segments of DNA that can jump from one position to another in the genome of a single cell. For this movement to occur, the transposon codes for a protein called the transposase that catalyses the transfer of the DNA from the donor location to a new randomly selected target location. The subject of this study, the IS608 transposon from the gastric pathogen Helicobacter pylroi is, however, unique in this regard. It only deals with a single strand of DNA throughout its pathway and inserts specifically at a ‘TTAC’ nucleotide sequence. In the presented work, we show that the target specificity of IS608 can be further increased by simply modifying parts of the transposon sequence.

The transposase of IS608, called TnpA employs a very elegant mechanism to cut and insert the transposon sequence into the DNA. The enzyme co-opts a part of the transposon DNA, which then recognizes the ‘TTAC’ sequence to which the transposon must join through base-pairing (Figure 1). Previous studies have shown that by changing this segment of the transposon DNA, the element can be driven to insert at sites other than TTAC. This opened up the possibility, to retarget the IS608 transposon to novel sites without changing any part of the protein TnpA. This marked a change in paradigm within the existing strategies to retarget transposons.

Can IS608 be targeted to unique genomic sequences?
The need of the hour in genetic tools is a system that can insert genetic information with site specificity. IS608 could be the answer if it could be redirected to longer sequences that are unique in the genome. We show that this is indeed possible. Our in vitro activity assays demonstrate that IS608 can be retargeted specifically to several unique sequences by modifying DNA sequences at the left end (LE) of the transposon. The implemented modifications were designed as to allow more specific base-pairings between the transposon and target DNA, thereby increase its selectivity (Figure 1). We are now carrying out in vivo assays in Escherichia coli to confirm that our IS608 retargeting design also works in a living system.

The mechanism of the IS608 jumping has been quite well characterized in the past. Therefore, it was a great surprise that our assays also identified an additional nucleotide that is essential for efficient transposition. By using X-ray crystallography, we hope to be able to explain how this nucleotide might be promoting the activity of the transposon.

Together, our results demonstrate that IS608 can be specifically directed to longer sequences, and also shed light on an additional level of complexity present in its transposition mechanism. This study also serves as a starting point to investigate if single-stranded DNA transposons such as IS608 can be developed as programmable genetic tools to take our DNA of interest as cargo and insert it into the specific location in the genome that we desire.

Advisor: Orsolya Barabas
Master’s Degree Project in Molecular Biology 30 credits 2015
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Kumar, Banushree
supervisor
organization
course
MOBM01 20151
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
7861784
date added to LUP
2015-09-08 14:53:20
date last changed
2015-09-08 14:53:20
@misc{7861784,
  abstract     = {DNA transposons are a class of mobile genetic elements that can autonomously move from one genomic location to another. They are powerful drivers of genetic change and have played a significant role in the evolution of many genomes. One such transposable element, IS608 from Helicobacter pylori employs a unique mechanism of transposition as it transposes in a single-stranded DNA form and inserts specifically 3’ of a specific tetranucleotide sequence (Kersulyte et al., 2002; Guynet et al., 2008). Previous structural (Ronning et al., 2005; Barabas et al, 2008) and biochemical studies (Ton-Hoang et al., 2005) of IS608, revealed that the element chooses its integration site specifically via base-pairing between the transposon and the target DNA. This unique feature allowed re-directing transposon integration to various four-nucleotide sequences by simply modifying the transposon DNA sequence. A key feature of the retargeting strategy was that, unlike the previous attempts to retarget mobile elements, the target specificity of IS608 could be varied straightforwardly by altering the transposon sequence without any need to modify the transposase protein. 
In this study, I tested if the target selection mechanism of IS608 could be exploited to direct the transposon specifically to longer DNA sequences, which could potentially be unique in the context of a genome. To this end, I first used an oligonucleotide based retargeting assay in vitro, where both the transposon and target sequences were engineered to allow specific selection of 12-nucleotide target sequences. The results of this assay showed that IS608 can be retargeted in a programmable way to multiple sequences in vitro. Additionally, the activity and selectivity of transposon integration could be further improved by the presence of a specific nucleotide adjacent to the TTAC in the target sequence, which revealed a novel feature of the IS608 transposition mechanism. Currently, we are pursuing crystallization of IS608 transposase-transposon-target DNA complexes that will further elucidate the molecular principles of this finding. Candidate transposon sequence variants were taken forward to an in vivo mating out assay in E. coli. In this ongoing assay, the rates of transposon integration at specific target sequences will be calculated and compared to the integration at non-specific targets, giving an estimate of the retargeting efficiency. In summary, a combination of molecular biology techniques, including structural biology, biochemistry, and microbiology, allowed me to contribute to the body of knowledge regarding IS608’s integration mechanism, which will hopefully in the future allow the development of a new, programmable tool for functional genomics, synthetic biology, and medicinal genetics.},
  author       = {Kumar, Banushree},
  language     = {eng},
  note         = {Student Paper},
  title        = {Retargeting of the IS608 Transposon},
  year         = {2015},
}