Expression, purification and preliminary crystallographic studies of a single-point mutant of Mos1 mariner transposase

Richardson, Julia M.; Zhang, Lei; Marcos, Séverine; Finnegan, David J.; Harding, Marjorie M.; Taylor, Paul; Walkinshaw, Malcolm D.

doi:10.1107/s0907444904003798

Cited by 18 publications

(26 citation statements)

References 15 publications

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“…This is probably partly due to an underestimation of the complexity of synaptic complexes. However, before we can access significant new crystallographic data it will be necessary, firstly, to control the proteolysis of these proteins during their production (Richardson et al 2004;Hickman et al 2005;Richardson et al 2006) and secondly, to distinguish between the various conformers that occur in a mix of purified transposase (Garcia-Saez and .…”

Section: Discussionmentioning

confidence: 99%

Assembly of the Tc1 and mariner transposition initiation complexes depends on the origins of their transposase DNA binding domains

2006

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In this review, we focus on the assembly of DNA/protein complexes that trigger transposition in eukaryotic members of the IS630-Tc1-mariner (ITm) super-family, the Tc1- and mariner-like elements (TLEs and MLEs). Elements belonging to this super-family encode transposases with DNA binding domains of different origins, and recent data indicate that the chimerization of functional domains has been an important evolutionary aspect in the generation of new transposons within the ITm super-family. These data also reveal that the inverted terminal repeats (ITRs) at the ends of transposons contain three kinds of motif within their sequences. The first two are well known and correspond to the cleavage site on the outer ITR extremities, and the transposase DNA binding site. The organization of ITRs and of the transposase DNA binding domains implies that differing pathways are used by MLEs and TLEs to regulate transposition initiation. These differences imply that the ways ITRs are recognized also differ leading to the formation of differently organized synaptic complexes. The third kind of motif is the transposition enhancers, which have been found in almost all the functional MLEs and TLEs analyzed to date. Finally, in vitro and in vivo assays of various elements all suggest that the transposition initiation complex is not formed randomly, but involves a mechanism of oriented transposon scanning.

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Section: Discussionmentioning

confidence: 99%

Assembly of the Tc1 and mariner transposition initiation complexes depends on the origins of their transposase DNA binding domains

2006

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“…Active mariner transposons have been tested for vertebrate transgenesis Mos1 and Himar1 have been widely studied using in vitro experiments dealing with biochemical transposition mechanisms (Augé-Gouillou et al 2001a, b, 2005aDawson and Finnegan 2003;Richardson et al 2004Richardson et al , 2006Richardson et al , 2007Lampe et al 1996Lampe et al , 1998Lampe et al , 1999. Both Mos1 and Himar1 purified transposases have been demonstrated to catalyse in vitro transposition (Lampe et al 1996;Tosi and Beverley 2000).…”

Section: Some Mariner Transposons Are Autonomous Elementsmentioning

confidence: 98%

Mariner transposons as genetic tools in vertebrate cells

et al. 2009

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Transposable elements (TEs) are being investigated as potential molecular tools in genetic engineering, for use in procedures such as transgenesis and insertional mutagenesis. Naturally active and reconstructed active TEs are both being studied to develop non-viral delivery vehicles. To date, the active elements being used include three Mariner-Like Elements (MLEs). We review below the studies that have investigated the ability of these MLEs to insert a transgene in vertebrate cells.

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“…Hyperactive single MOS1 mutants with altered protein-protein interactions Richardson et al (2004) has identified a single-point MOS1 mutant (T216A) from a yeast two-hybrid assay designed to identify transposase mutants that are able to interact more strongly with the wild-type protein. Recently, T216A was found to dimerize more in solution than MOS1[?]…”

Section: Single Mos1 Mutants Containing Mutations Previously Charactementioning

confidence: 99%

Mariner Mos1 transposase optimization by rational mutagenesis

et al. 2009

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Mariner transposons are probably the most widespread transposable element family in animal genomes. To date, they are believed not to require species-specific host factors for transposition. Despite this, Mos1, one of the most-studied mariner elements (with Himar1), has been shown to be active in insects, but inactive in mammalian genomes. To circumvent this problem, one strategy consists of both enhancing the activity of the Mos1 transposase (MOS1), and making it insensitive to activity-altering post-translational modifications. Here, we report rational mutagenesis studies performed to obtain hyperactive and non-phosphorylable MOS1 variants. Transposition assays in bacteria have made it possible to isolate numerous hyperactive MOS1 variants. The best mutant combinations, named FETY and FET, are 60- and 800-fold more active than the wild-type MOS1 version, respectively. However, there are serious difficulties in using them, notably because they display severe cytotoxicity. On the other hand, three positions lying within the HTH motif, T88, S99, and S104 were found to be sensitive to phosphorylation. Our efforts to obtain active non-phosphorylable mutants at S99 and S104 positions were unsuccessful, as these residues, like the co-linear amino acids in their close vicinity, are critical for MOS1 activity. Even if host factors are not essential for transposition, our data demonstrate that the host machinery is essential in regulating MOS1 activity.

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Expression, purification and preliminary crystallographic studies of a single-point mutant of Mos1 mariner transposase

Cited by 18 publications

References 15 publications

Assembly of the Tc1 and mariner transposition initiation complexes depends on the origins of their transposase DNA binding domains

Assembly of the Tc1 and mariner transposition initiation complexes depends on the origins of their transposase DNA binding domains

Mariner transposons as genetic tools in vertebrate cells

Mariner Mos1 transposase optimization by rational mutagenesis

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