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A series of constitutive expression vectors to accurately measure the rate of DNA
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transposition and correct for auto-inhibition
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Michael Tellier* and Ronald Chalmers*
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School of Life Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham,
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NG7 2UH, UK
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* Correspondence: michael.tellier@path.ox.ac.uk; ronald.chalmers@nottingham.ac.uk
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Present Address: Michael Tellier, Sir William Dunn School of Pathology, University of
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Oxford, Oxford, OX1 3RF, UK
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.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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Abstract
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Background
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Transposable elements (TEs) form a diverse group of DNA sequences encoding functions
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for their own mobility. This ability has been exploited as a powerful tool for molecular biology
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and genomics techniques. However, their use is sometimes limited because their activity is
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auto-regulated to allow them to cohabit within their hosts without causing excessive genomic
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damage. To overcome these limitations, it is important to develop efficient and simple
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screening assays for hyperactive transposases.
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Results
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To widen the range of transposase expression normally accessible with inducible promoters,
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we have constructed a set of vectors based on constitutive promoters of different strengths.
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We characterized and validated our expression vectors with Hsmar1, a member of the
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mariner transposon family. We observed the highest rate of transposition with the weakest
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promoters. We went on to investigate the effects of mutations in the Hsmar1 transposase
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dimer interface and of covalently linking two transposase monomers in a single-chain dimer.
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We also tested the severity of mutations in the lineage leading to the human SETMAR gene,
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in which one copy of the Hsmar1 transposase has contributed a domain.
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Conclusions
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We generated a set of vectors to provide a wide range of transposase expression which will
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be useful for screening libraries of transposase mutants. We also found that mutations in the
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Hsmar1 dimer interface provides resistance to overproduction inhibition in bacteria, which
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could be valuable for improving bacterial transposon mutagenesis techniques.
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.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 18, 2019. ; https://doi.org/10.1101/423012doi: bioRxiv preprint
3
Keywords
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Papillation assay, Hsmar1, overproduction inhibition, SETMAR, transposase, transposable
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elements.
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.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 18, 2019. ; https://doi.org/10.1101/423012doi: bioRxiv preprint
4
Background
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Transposable elements (TEs) are DNA sequences encoding their own ability to move in a
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genome from one place to another. They are found in virtually all organisms and are
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particularly present in eukaryotes where they can represent a high percentage of the
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genome (1-3). Originally described as selfish elements since they were considered parasites
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which use the host for propagation but do not provide any particular advantage, TEs have
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now been shown to be important drivers of genome evolution (4, 5). Indeed, TEs can provide
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novel transcription factor binding sites, promoters, exons or poly(A) sites and can also be co-
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opted as microRNAs or long intergenic RNAs (6-8). TEs are a diverse group of DNA
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sequences using a wide range of mechanisms to transpose within their hosts. One particular
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mechanism prevalent in eukaryotes, and used by the mariner family, is known as “cut-and-
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paste” transposition (9). Over the past several years, our group and others have described
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the mechanisms regulating the transposition rate of different mariner transposons, such as
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Himar1, Hsmar1 or Mos1 (10-15). In Hsmar1, a regulatory mechanism was first recognized
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because of the phenomenon of overproduction inhibition (OPI) (16). The mechanism of OPI
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was eventually explained by the realization that double occupancy of the transposon ends
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with transposase dimers blocks assembly of the transpososome (12). Thus, OPI curbs
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Hsmar1 transposition rate to avoid damaging the host genome by excessive transposition
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(12).
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However, OPI represents a limitation in the development of hyperactive transposases, which
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would facilitate transposon mutagenesis. Several approaches such as modifying the binding
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kinetics of the transposase to the inverted terminal repeat (ITR) or the monomer-dimer
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equilibrium can be used to overcome OPI. Indeed, we and others previously showed that
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most mutations in the conserved motif, WVPHEL, in Himar1 and Hsmar1, located at the
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subunit interface, result in hyperactive transposases but at the cost of producing non-
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productive DNA double-strand breaks and therefore DNA damage (17, 18).
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.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 18, 2019. ; https://doi.org/10.1101/423012doi: bioRxiv preprint
5
To facilitate the isolation of suitable transposase mutants, the papillation assay was
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developed as an efficient screening procedure (Supplementary Figure 1) (19, 20). This
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assay is based on a promoter-less lacZ gene flanked by transposon ends. This reporter is
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integrated in a silent region of the genome of Escherichia coli. The transposase gene is
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provided in trans on a plasmid to simplify mutagenesis and library handling. Transposition
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events into an expressed ORF give rise to lacZ gene fusion proteins. When this happens
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within a colony growing on an X-gal indicator plate, it converts the cell to a lac+ phenotype,
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which allows the outgrowth of blue microcolonies (papillae) on a background of white cells.
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The transposition rate is estimated by the number of papillae per colony and by the rate of
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their appearance.
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A limitation of the papillation assay is that it generally employs a transposase gene whose
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expression is under the control of an inducible promoter which cannot be finely regulated.
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We have constructed a set of vectors maintained in single copy or as five copies per cell
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which carry various constitutive promoters in the absence or presence of a ribosome binding
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site (RBS). This set of vectors allows transposase expression across a wide range of
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expression levels facilitating the screening of hyperactive and/or OPI-resistant transposases.
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We used this set of vectors to compare an Hsmar1 transposase monomer to a single-chain
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dimer and to test for hyperactivity and OPI-resistance several Hsmar1 transposase mutants.
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We found that one Hsmar1 mutant in the dimer interface, R141L, is resistant to OPI in E. coli.
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Results and Discussion
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Characterization of the papillation assay using a strong inducible promoter
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The papillation assay provides a visual assessment of the transposition rate, which can be
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determined from the rate of papillae appearance and their number per colony (19). The
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transposition rate is dependent on the concentration and activity of the transposase (12). We
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defined the transposition rate as the average number of papillae per colony after five days of
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.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 18, 2019. ; https://doi.org/10.1101/423012doi: bioRxiv preprint
