Early Intermediates of mariner Transposition: Catalysis without Synapsis of the Transposon Ends Suggests a Novel Architecture of the Synaptic Complex

Abstract

The mariner family is probably the most widely distributed family of transposons in nature. Although these transposons are related to the well-studied bacterial insertion elements, there is evidence for major differences in their reaction mechanisms. We report the identification and characterization of complexes that contain the Himar1 transposase bound to a single transposon end. Titrations and mixing experiments with the native transposase and transposase fusions suggested that they contain different numbers of transposase monomers. However, the DNA protection footprints of the two most abundant single-end complexes are identical. This indicates that some transposase monomers may be bound to the transposon end solely by protein-protein interactions. This would mean that the Himar1 transposase can dimerize independently of the second transposon end and that the architecture of the synaptic complex has more in common with V(D)J recombination than with bacterial insertion elements. Like V(D)J recombination and in contrast to the case for bacterial elements, Himar1 catalysis does not appear to depend on synapsis of the transposon ends, and the single-end complexes are active for nicking and probably for cleavage. We discuss the role of this single-end activity in generating the mutations that inactivate the vast majority of mariner elements in eukaryotes.The Tc1/mariner superfamily of transposons consists of the Tc1 and mariner families of elements in eukaryotes and the more distantly related IS630-like elements in bacteria (25, 39). Members of the superfamily have a single transposase gene expressed in the germ line and/or the soma, transpose via a "cut and paste" DNA intermediate, and duplicate a TA dinucleotide upon insertion. This is probably the most widespread family of transposons in nature: members have been identified in bacteria (IS630), ciliates, fungi, plants, and most animal phyla, from Porifera (sponges) to humans. Although mariner elements are widespread, they are unevenly distributed in closely related species and the vast majority are inactive because of mutations. This suggests that they have an unusual life style that involves a high rate of horizontal transfer to new hosts, followed by a burst of transposition and subsequent vertical inactivation (37, 43).Homology-dependent gene silencing serves to control the spread of transposons, retroviruses, and other repetitive DNA elements in many eukaryotes. It is mediated by at least three distinct mechanisms, specifically DNA methylation, histone deacetylation, and RNA interference. However, depending on the identity of the organism and the repetitive element in question, these mechanisms are not always completely effective. As noted by McClintock, the rate of transposition in a given germ line can change over time, with cycles of activation and silencing lasting several generations (cited in reference 35). Furthermore, although all three mechanisms of homologydependent gene silencing operate in plants and are active against many retrotransposons, som...

Assembly of the mariner Mos1 Synaptic Complex

“…6, lane 6, in reference 9), which is in agreement with the data presented here. Finally, similar results were reported for the Himar1 transposon (18). Cleavage sites observed in PEC2 were defined in the same way as for SEC2 and PEC1.…”

“…This configuration would allow the Tnp bound to the ITR to be arranged side by side, but facing in opposite directions, on the same side of the DNA double helix. This proposal is sustained by the recent work of Lipkow et al (18). They have described two patches of the Himar1 ITR protected by the Tnp and centered on positions 10 and 20.…”

“…The same observations have been carried out for V(D)J recombination, where SEC1 and SEC2 undergo single-strand nicking in MgCl 2 (20). The ability of SEC2 to undergo cleavages (9,18; this study) is unexpected, because it provides an opportunity for deleterious breakage to occur as a result of protein assembly on a single ITR. In many transposition pathways, this is prevented by the fact that both DNA ends must be present before the proteins required for catalysis can be assembled.…”

“…Despite the recent description of the cleavages occurring during excision (9), little is known about the formation of synaptic complexes in mariner transposons. Indeed, in a recent publication, Lipkow et al (18) failed to observe the Himar1 synaptic complex, thus preventing the characterization of this complex, even if they were able to detect its activity. Here, we determined the Tnp content and functional capabilities of Mos1 Tnp/ITR complexes.…”

A Simple Topological Filter in a Eukaryotic Transposon as a Mechanism To Suppress Genome Instability

“…This was supported by several lines of biochemical evidence that have, until now, been generally accepted. These observations form the basis for the current model for mariner transposition, which postulates that the first nick is followed by synapsis, which induces a conformational change, or subunit exchange, that licenses secondstrand cleavage (13,19). This model is contradicted by the experiments with the single-ended plasmid (Fig.…”