Mariner Mos1 Transposase Dimerizes Prior to ITR Binding Corinne Auge ´ -Gouillou * , Benjamin Brillet, Ste ´ phanie Germon Marie-He ´le ` ne Hamelin and Yves Bigot * Laboratoire d’Etude des Parasites Ge ´ne ´tiques, Universite ´ Franc ¸ois Rabelais de Tours EA 3868, UFR Sciences & Techniques, Parc Grandmont 37200 Tours, France The mariner Mos1 synaptic complex consists of a tetramer of transposase molecules that bring together the two ends of the element. Such an assembly requires at least two kinds of protein–protein interfaces. The first is involved in “cis” dimerization, and consists of transposase molecules bound side-by-side on a single DNA molecule. The second, which is involved in “trans” dimerization, consists of transposase molecules bound to two different DNA molecules. Here, we used biochemical and genetic methods to enhance the definition of the regions involved in cis and trans-dimerization in the mariner Mos1 transposase. The cis and trans-dimerization interfaces were both found within the first 143 amino acid residues of the protein. The cis-dimerization activity was mainly contained in amino acids 1–20. The region spanning from amino acid residues 116–143, and containing the WVPHEL motif, was involved in the cis- to trans-shift as well as in trans-dimerization stabilization. Although the transposase exists mainly as a monomer in solution, we provide evidence that the transposase cis-dimer is the active species in inverted terminal repeat (ITR) binding. We also observed that the catalytic domain of the mariner Mos1 transposase modulates efficient transposase– transposase interactions in the absence of the transposon ends. q 2005 Elsevier Ltd. All rights reserved. Keywords: mariner; synaptic complex; transposase–transposase interfaces; dimerization; tetramerization *Corresponding authors Introduction Nucleoprotein complexes play a central role in all aspects of genetic activity, including transcription, packaging, rearrangement, replication and repair. Complexes involved in the mobility of transposable elements have been extensively explored in pro- karyote cells. Less is known about the organization and function of synaptic complexes of eukaryotic elements, such as those of the mariner-like element (MLE) family, that occur naturally in a wide range of eukaryotes, 1 including human beings. 2,3 One of the MLEs, the autonomous Mos1 element, is 1286 bp long, with 28 bp, imperfect, inverted terminal repeats (ITRs), and contains a single open reading frame coding a 345 amino acid transposase (Tnp). We have recently shown 4 that the mariner Mos1 synaptic complex consists of a tetramer of transposases that brings together the two ITRs. The assembly of the synaptic complex is an obligatory step in the “excision–reinsertion” transposition pathway of the MLEs, as well as of several other class II elements. 5 It is therefore important to investigate the organization of such complexes, as this is the basis of our understanding of how MLE transposition works. Experimental data about mariner transposase activities, 6,7 combined with computer analyses, 8 have made it possible to describe this protein as being organized in two main domains (Figure 1(a)). The N-terminal domain of the transposase (amino acid residues 1–119) contains a helix-turn-helix (HTH) motif, which is known to be involved in ITR binding. To date, no function has been assigned to the region that spans from amino acid 119–156, 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: ITR, inverted terminal repeat; MLE, mariner-like element; Tnp, transposase; WT, wild- type; HIV-1, human immunodeficiency virus type-1; HTH, helix-turn-helix; ESMA, electrophoretic mobility shift assay; aa, amino acid residue; SB, Sleeping Beauty; MBP, maltose binding protein. E-mail addresses of the corresponding authors: auge@univ-tours.fr; bigot@univ-tours.fr doi:10.1016/j.jmb.2005.05.019 J. Mol. Biol. (2005) 351, 117–130