Abstract:Background:The target capture is a critical step for the selection of integration site. Results: Mos1 transposon excision occurred before target capture. The TA dinucleotide present in the target and bent targets are important for this step. Conclusion: Target capture mechanism and distribution of mariner elements are linked. Significance: New insights for modeling Mos1 target capture complex and better understanding of mariner transposition cycle.
“…Target DNA binds in a channel between the two catalytic domains and the active sites contain the strand transfer products. As the TCC also contains a transposase dimer (Pflieger et al, 2014), our new STC structure indicates that Mos1 strand transfer, like transposon excision, is catalysed by a transposase dimer.10.7554/eLife.15537.006Figure 2.Architecture of the Mos1 strand transfer complex.( a ) Structure of the STC, with transposase subunits (orange and blue), IR DNA (orange and green) and target DNA (magenta and black). Figure 2—figure supplement 1 shows the crystal packing arrangement.…”
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
“…Target DNA binds in a channel between the two catalytic domains and the active sites contain the strand transfer products. As the TCC also contains a transposase dimer (Pflieger et al, 2014), our new STC structure indicates that Mos1 strand transfer, like transposon excision, is catalysed by a transposase dimer.10.7554/eLife.15537.006Figure 2.Architecture of the Mos1 strand transfer complex.( a ) Structure of the STC, with transposase subunits (orange and blue), IR DNA (orange and green) and target DNA (magenta and black). Figure 2—figure supplement 1 shows the crystal packing arrangement.…”
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
“…Our analyses were done using two sources of recombinant MBP-MOS1: one was expressed in a prokaryote (E. coli, JM109 strain, with the pMAL-New expression plasmid; New England Biolabs) because all the biochemical studies performed to date used this, and the other was expressed in a eukaryote (sf21 insect cells, with the pVL1392 baculovirus transfer vector and the BaculoGold baculovirus expression system, BD Biosciences) [31] because insects are actually the natural hosts of Mos1. The fusion proteins were purified on an amylose-binding resin (New England Biolabs), as previously described.…”
Section: Resultsmentioning
confidence: 99%
“…Thus, only the height (z) is considered. The central region is described by two large peaks, each of 70-80 , whereas the monomer has one 70-80 peak and two smaller peaks (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). According to what we know about MOS1 dimerisation (at the N-termini, that is, the first HTH) [4,6] and the position of the MBP in the fusion protein (Nterminal fusion), we assume that, for each monomer, the large blue/white dome (70-80 ) corresponds to the MBP and the first HTH of the transposase; the two smaller domains could be the second HTH and the MOS1 catalytic domain (linked by a flexible linker; Figure 4 C).…”
Transposases are specific DNA-binding proteins that promote the mobility of discrete DNA segments. We used a combination of physicochemical approaches to describe the association of MOS1 (an eukaryotic transposase) with its specific target DNA, an event corresponding to the first steps of the transposition cycle. Because the kinetic constants of the reaction are still unknown, we aimed to determine them by using quartz crystal microbalance on two sources of recombinant MOS1: one produced in insect cells and the other produced in bacteria. The prokaryotic-expressed MOS1 showed no cooperativity and displayed a Kd of about 300 nM. In contrast, the eukaryotic-expressed MOS1 generated a cooperative system, with a lower Kd (∼ 2 nm). The origins of these differences were investigated by IR spectroscopy and AFM imaging. Both support the conclusion that prokaryotic- and eukaryotic-expressed MOS1 are not similarly folded, thereby resulting in differences in the early steps of transposition.
“…185 Another feature of target sites that has been reported is a preference for “bendable” sites. 157–160 Indeed, the MuA transposase preferentially and specifically targets DNA containing mismatched base pairs, most likely due to structural deformability of the target DNA. 186 Genomic context can also be important, as some transposons exhibit a preference for insertion into promoter regions or transcriptional start sites, others for genes or transcriptional units.…”
Section: Transposons As Genetic Tools: a Versatile Toolsetmentioning
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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