Towards an in vitro recombination system mediated by the maize Activator (Ac) element transposase
--Rudenko, GN and Kunze, R

Transposition of Ac is mediated in vivo by the element-encoded transposase, a protein of 807 AA with a molecular weight of 112 kD. Ac is structurally similar to some other eukaryotic transposable elements. These elements generate 8 bp duplications at their genomic integration sites and the sequences of their terminal inverted repeats are similar. The polypeptide sequences of the TPases of these elements are highly homologous along their ca. 600 C-terminal residues. This suggests a common mechanism of transposition. It is believed that Ac transposition occurs in a non-replicative manner via a "cut-and-paste" mechanism similar to that of the P element from Drosophila and bacterial transposons Tn7 and Tn10. Genetic data indicate an association of Ac transposition with DNA replication. However, no in vitro transposition products or reaction intermediates involving eukaryotic transposases have been described until now.

To study the activities of the Ac TPase on the enzymatic level, two interrelated aspects are being approached. One concerns the identification of Ac TPase enzymatic activities and the biochemistry of specific TPase-mediated DNA rearrangements. Localization, mapping and further dissection of the specific Ac TPase catalytic domain(s) responsible for the recombination reactions is the second scope.

To begin with dissection of the components required for a cell-free transposition system, we have primarily concentrated our attention on the wild type Ac TPase (1-807 AA) and its N-terminally truncated derivative (103-807 AA). Both proteins are functional in vivo and recognize in vitro specifically the 11 bp terminal inverted repeats of the element and multiple AAACGG or similar sequence motifs present in its subterminal regions. These proteins as well as a number of mutant derivatives were overexpressed in E.coli cells and purified either using Ni-chelate affinity chromatography and gel filtration on Superdex 200 column and/or by preparative SDS-polyacrylamide electrophoresis. Final preparations are free of contaminating proteins as judged by Western blot analysis and visual inspection of protein gels. Purified proteins have been tested for DNA-binding activity using gel-retardation assays.

It is important to note that all recombinases studied to date have a DNA-topoisomerase activity. Association of a topoisomerase-like activity with Ac TPase might be a key to our understanding of the Ac TPase functionality. Therefore purified TPase preparations have been tested for relaxation activity in standard assays using either a negatively supercoiled substrate DNA construct containing a complete Ds element (a non-autonomous Ac derivative) or fc 174 DNA as a control.  We have been able to detect such an activity for the wild-type transposase and a number of its derivatives.  The relaxation activity of the TPase is ATP-independent.  It is not stimulated by additions of mono-, divalent (except Mg) cations and spermidine.  Preliminarily the protein can be classified as a type I DNA-topoisomerase.

The topoisomer pattern generated by the TPase on transposon- containing DNA is however qualitatively different from the one obtained for fc 174 DNA.  Under conditions when fc 174 DNA is fully relaxed, transposon-containing DNA always remains underrelaxed.  The protection of some supercoils from the topoisomerase activity indicates a different mode of interaction between TPase and a substrate DNA depending on the presence or absence of a transposable element in its context.

To study TPase-DNA interactions in more detail we have used glass-fiber filters to selectively bind DNA-protein complexes out of reaction mixtures. This allows separation from free DNA which is not retained on the filter. In the absence of divalent cations strong binding of the TPase could be detected not only to the Ds-containing plasmid DNA but also to single- and double-stranded fc 174 DNAs.  Selectivity of TPase binding toward Ds containing DNA can however be induced by addition of divalent cations (Mg or Ca). Under these conditions TPase does not bind to either of the fc 174 DNAs.  The nucleoprotein complex formed between TPase and DNA is also quite unusual since it can be dissociated only by a treatment with protein denaturants. Comparative studies made on linear or circular DNA substrates lead us to a preliminary conclusion about the existence of a topological lock between TPase and DNA in the form of protein clamp around DNA.

Experiments are under way to determine in which way the structural features of TPase-DNA complexes and a topoisomerase activity displayed by the TPase could be involved in transpositional recombination. 

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