Structural Basis for the Inverted Repeat Preferences of mariner Transposases

Trubitsyna, Maryia; Grey, Heather; Houston, Douglas R.; Finnegan, David J.; Richardson, Julia M.

doi:10.1074/jbc.m115.636704

Cited by 9 publications

(11 citation statements)

References 40 publications

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“…The transposon containing the kanamycin resistance cassette only was constructed previously ( 38 ). We used donor plasmids with either two right Mos1 IRs (pEPMosRR) or with two left Mboumar-9 IRs (pEPMboLL) as they were shown to be the most efficient for in vitro transposition ( 38 ). The SalI site within the backbone pEP185.2 was deleted by site directed mutagenesis using primers 5΄-ccctcgaggtagacggtatcgataagc-3΄ and 5΄-gcttatcgataccgtctacctcgaggg-3΄.…”

Section: Methodsmentioning

confidence: 99%

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection

Trubitsyna

Michlewski

Finnegan

et al. 2017

Nucleic Acids Research

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Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing.

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Section: Methodsmentioning

confidence: 99%

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection

Trubitsyna

Michlewski

Finnegan

et al. 2017

Nucleic Acids Research

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“…Nonidentical transposon ends often correlate to differences in transposase binding affinity. It has been shown that modifying Mos1 (with its asymmetric IRL/IRR ends) such that it has two identical IRR/IRR ends increases the frequency of in vitro transposition ~26-fold, 147 correlating to the reported tighter binding of the Mos1 transposase to its IRR sequence than to its IRL. 149…”

Section: Insight Into Dna Transposition Mechanisms From Structure: Ovmentioning

confidence: 99%

DNA Transposition at Work

Hickman

Dyda

2016

Chem. Rev.

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DNA transposons are defined segments of DNA that are able to move from one genomic location to another. Movement is facilitated by one or more proteins, called the transposase, typically encoded by the mobile element itself. Here, we first provide an overview of the classification of such mobile elements in a variety of organisms. From a mechanistic perspective, we have focused on one particular group of DNA transposons that encode a transposase with a DD(E/D) catalytic domain that is topologically similar to RNase H. For these, a number of three-dimensional structures of transpososomes (transposase-nucleic acid complexes) are available, and we use these to describe the basics of their mechanisms. The DD(E/D) group, in addition to being the largest and most common among all DNA transposases, is the one whose members have been used for a wide variety of genomic applications. Therefore, a second focus of the article is to provide a nonexhaustive overview of transposon applications. Although several non-transposon-based approaches to site-directed genome modifications have emerged in the past decade, transposon-based applications are highly relevant when integration specificity is not sought. In fact, for many applications, the almost-perfect randomness and high frequency of integration make transposon-based approaches indispensable.

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“…The binding occurs at the DNA binding domain (DBD) of the transposase. Since TIR sequences can vary at each end, the transposase binding has different affinities to these sequences [20]. For instance, the PBE of the rice MLE, Osmar5 is 17 bp long and composed of two boxes, Box I and Box II.…”

Section: Introductionmentioning

confidence: 99%

“…In contrast, when the left TIR was replaced by the right, the affinity was found to shift in the opposite direction by showing an increase by more than 26 times [21,22,23]. Likewise, when the guanines of 3′ TIRs of the ant ( Messor bouvieri ) transposon, Mboumar9 were replaced by adenines, the TIR affinity was found four times higher [20]. Additionally, the subterminal region, 30–35 bp flanking sequences of the TIR (Sub-TIR) can also affect the transposition efficiency.…”

Section: Introductionmentioning

confidence: 99%

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Affinities of Terminal Inverted Repeats to DNA Binding Domain of Transposase Affect the Transposition Activity of Bamboo Ppmar2 Mariner-Like Element

Ramakrishnan

Zhou

Pan

et al. 2019

IJMS

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Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2, have previously been identified in moso bamboo (Phyllostachys edulis). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR’s affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5–2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2. The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies.

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Structural Basis for the Inverted Repeat Preferences of mariner Transposases

Cited by 9 publications

References 40 publications

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection

DNA Transposition at Work

Affinities of Terminal Inverted Repeats to DNA Binding Domain of Transposase Affect the Transposition Activity of Bamboo Ppmar2 Mariner-Like Element

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