Abstract:Most DNA transposons move from one
genomic location to another
by a cut-and-paste mechanism and are useful tools for genomic manipulations.
Short inverted repeat (IR) DNA sequences marking each end of the transposon
are recognized by a DNA transposase (encoded by the transposon itself).
This enzyme cleaves the transposon ends and integrates them at a new
genomic location. We report here a comparison of the biophysical and
biochemical properties of two closely related and active mariner/Tc1 family DNA transposa… Show more
“…The classic problem of inclusion body aggregation in overexpression in E. coli [31] was overcome by a combination of low temperature incubation as shown for TPases of transposons such as Ac, Tol2, Mos1 and Mboumar-9 [25,[33][34][35], and early log-phase induction as previously described [32]. In our study, the percentage of expressed soluble protein increased with a concomitant reduction of endogenous host proteins making for a more efficient purification process.…”
Goldfish Tgf2 transposon of Hobo/Activator/Tam3 (hAT) family can mediate gene insertion in a variety of aquacultural fish species by transposition; however, the protein structure of Tgf2 transposase (TPase) is still poorly understood. To express the goldfish Tgf2 TPase in Escherichia coli, the 2061-bp coding region was cloned into pET-28a(+) expression vector containing an N-terminal (His)6-tag. The pET-28a(+)-Tgf2 TPase expression cassette was transformed into Rosetta 1 (DE3) E. coli lines. A high yield of soluble proteins with molecular weight of ~80 kDa was obtained by optimized cultures including low-temperature (22 °C) incubation and early log phase (OD600 = 0.3-0.4) induction. Mass spectrometry analysis following trypsin digestion of the recombinant proteins confirmed a Tgf2 TPase component in the eluate of Ni(2+)-affinity chromatography. When co-injected into 1-2 cell embryos with a donor plasmid harboring a Tgf2 cis-element, the prokaryotic expressed Tgf2 TPase can mediate high rates (45 %) of transposition in blunt snout bream (Megalobrama amblycephala). Transposition was proved by the presence of 8-bp random direct repeats at the target sites, which is the signature of hAT family transposons. Production of the Tgf2 Tpase protein in a soluble and active form not only allows further investigation of its structure, but provides an alternative tool for fish transgenesis and insertional mutagenesis.
“…The classic problem of inclusion body aggregation in overexpression in E. coli [31] was overcome by a combination of low temperature incubation as shown for TPases of transposons such as Ac, Tol2, Mos1 and Mboumar-9 [25,[33][34][35], and early log-phase induction as previously described [32]. In our study, the percentage of expressed soluble protein increased with a concomitant reduction of endogenous host proteins making for a more efficient purification process.…”
Goldfish Tgf2 transposon of Hobo/Activator/Tam3 (hAT) family can mediate gene insertion in a variety of aquacultural fish species by transposition; however, the protein structure of Tgf2 transposase (TPase) is still poorly understood. To express the goldfish Tgf2 TPase in Escherichia coli, the 2061-bp coding region was cloned into pET-28a(+) expression vector containing an N-terminal (His)6-tag. The pET-28a(+)-Tgf2 TPase expression cassette was transformed into Rosetta 1 (DE3) E. coli lines. A high yield of soluble proteins with molecular weight of ~80 kDa was obtained by optimized cultures including low-temperature (22 °C) incubation and early log phase (OD600 = 0.3-0.4) induction. Mass spectrometry analysis following trypsin digestion of the recombinant proteins confirmed a Tgf2 TPase component in the eluate of Ni(2+)-affinity chromatography. When co-injected into 1-2 cell embryos with a donor plasmid harboring a Tgf2 cis-element, the prokaryotic expressed Tgf2 TPase can mediate high rates (45 %) of transposition in blunt snout bream (Megalobrama amblycephala). Transposition was proved by the presence of 8-bp random direct repeats at the target sites, which is the signature of hAT family transposons. Production of the Tgf2 Tpase protein in a soluble and active form not only allows further investigation of its structure, but provides an alternative tool for fish transgenesis and insertional mutagenesis.
“…The individual substitutions W159A, K190A or F187A also reduced the in vitro transposition efficiency to <20% that of T216A Mos1 transposase (Figure 5). 10.7554/eLife.15537.014Figure 5.Residues that stabilise the transposition product are required for efficient Mos1 transposition in vitro.Efficiencies of an in vitro Mos1 hop assay, performed using Mos1 transposase mutants and donor plasmids containing a kanamycin resistance gene flanked by Mos1 inverted repeats, as described previously (Trubitsyna et al, 2014). Excision of the IR-flanked gene from a circular plasmid by transposase, and its integration into a supercoiled target plasmid, results in transfer of the kanamycin resistance to the target plasmid.…”
Cut-and-paste DNA transposons of the mariner/Tc1 family are useful tools for genome engineering and are inserted specifically at TA target sites. A crystal structure of the mariner transposase Mos1 (derived from Drosophila mauritiana), in complex with transposon ends covalently joined to target DNA, portrays the transposition machinery after DNA integration. It reveals severe distortion of target DNA and flipping of the target adenines into extra-helical positions. Fluorescence experiments confirm dynamic base flipping in solution. Transposase residues W159, R186, F187 and K190 stabilise the target DNA distortions and are required for efficient transposon integration and transposition in vitro. Transposase recognises the flipped target adenines via base-specific interactions with backbone atoms, offering a molecular basis for TA target sequence selection. Our results will provide a template for re-designing mariner/Tc1 transposases with modified target specificities.DOI:
http://dx.doi.org/10.7554/eLife.15537.001
“…The T m for Mos1 transposase has been reported previously. 46 In this work, we determine the melting temperature for the original SB and its hyperactive version SB100X by extrinsic (ANS) and intrinsic (Tyr and Trp) fluorescence.…”
Section: Experimental Determination Of T Mmentioning
DD[E/D]-transposases catalyze the multistep reaction of cut-and-paste DNA transposition. Structurally, several DD[E/D]-transposases have been characterized, revealing a multi-domain structure with the catalytic domain possessing the RNase H-like structural motif that brings three catalytic residues (D, D, and E or D) into close proximity for the catalysis. However, the dynamic behavior of DD[E/D]-transposases during transposition remains poorly understood. Here, we analyze the rigidity and flexibility characteristics of two representative DD[E/D]-transposases Mos1 and Sleeping Beauty (SB) using the minimal distance constraint model (mDCM). We find that the catalytic domain of both transposases is globally rigid, with the notable exception of the clamp loop being flexible in the DNA-unbound form. Within this globally rigid structure, the central β-sheet of the RNase H-like motif is much less rigid in comparison to its surrounding α-helices, forming a cage-like structure. The comparison of the original SB transposase to its hyperactive version SB100X reveals the region where the change in flexibility/rigidity correlates with increased activity. This region is found to be within the RNase H-like structural motif and comprise the loop leading from beta-strand B3 to helix H1, helices H1 and H2, which are located on the same side of the central beta-sheet, and the loop between helix H3 and beta-strand B5. We further identify the RKEN214-217DAVQ mutations of the set of hyperactive mutations within the catalytic domain of SB transposase to be the driving factor that induces change in residue-pair rigidity correlations within SB transposase. Given that a signature RNase H-like structural motif is found in DD[E/D]-transposases and, more broadly, in a large superfamily of polynucleotidyl transferases, our results are relevant to these proteins as well. K E Y W O R D S distance constraint model, DNA transposon, dynamics, flexibility and rigidity, fluorescence, protein-DNA complex, transposase that includes enzymes involved in replication, recombination, DNA repair, splicing, transposition, RNA interference (RNAi), and CRISPR-Cas immunity. 15,16 The RNase H-like structural motif is schematically shown in Figure 1. It consists of a central β-sheet comprised of five β-strands (ordered 32 145 with the second β-strand B2 antiparallel to other strands) and three surrounding α-helices. Two of the three α-helices (H1 and H2) are inserted in the amino acid sequence between β-strands B3 and B4 and B4 and B5, respectively, and are located on one side of the β-sheet. The third α-helix (H3) is located on the opposite side of the β-sheet and either immediately follows β-strand B5 or is separated from it by inserted amino acid sequence of various length. The most abundant proteins in the RNHL superfamily are DD[E/D]-transposases. 15 Although there are significant differences in sequence and structure across the RNHL superfamily, the RNase H architecture represents a highly conserved core of the catalytic domain of RNHL proteins, suggesting...
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