Tn 5 Synaptic Complex Formation : Role of Transposase Residue W 450

Tn5 transposase (Tnp) is a member of the retroviral integrase superfamily of proteins. This superfamily includes other Tnp’s, as well as proteins such as human immunodeficiency virus type 1 integrase and the RAG-1 recombinase of V(D)J recombination. The members of this superfamily share many mechanistic details, as well as structurally conserved active sites (4, 6, 12). In order to catalyze DNA movements, these proteins adopt highly ordered protein-DNA synaptic complexes. Based on structural studies with Tn5 Tnp, it is known that these complexes involve large numbers of protein-protein and protein-DNA contacts (3) and generally involve the rearrangement of the proteins. The catalytic steps of Tn5 transposition are depicted in Fig. 1. Tn5 transposition is a tightly regulated process, as excessive transposition activity would likely lead to instability of the host cell genome. Previous studies led to a model in which Tn5 Tnp monomers manifest an intramolecular interaction between the N terminus, containing end sequence (ES) DNA binding residues, and the C terminus, containing the protein-protein dimerization domain, that inhibits synaptic complex formation (9, 15). Tn5 Tnp is monomeric in solution and binds to ES DNA substrates only as a dimer (5, 9, 15). Tn5 inhibitor Inh (lacking 55 N-terminal residues) exists as a dimer in solution, suggesting that the N terminus of free Tnp blocks dimerization (9). When the C-terminal dimerization domain is removed from Tnp (in a C-terminal truncation mutant protein, 369), Tnp is unable to form dimers or synaptic complexes (14, 16, 17). However, 369 is able to bind to ES DNA as a monomer (17). Thus, the removal of the C terminus allows ES DNA binding. It is likely that C-terminal deletions of Mu and IS911 transposases also enhance ES DNA binding (1, 7). In this study, we investigated the role of amino acid W450 in transposition and demonstrated that this residue is inhibitory for synapsis and may contribute to the N terminus-C terminus inhibitory interaction. W450 is conserved across IS50/Tn5 transposase-related proteins (11). W450 is located at the beginning of the C-terminal -helix (3) that is spatially dislocated by the hyperactive L372P mutation (10, 15). W450 is also located adjacent to a previously identified, modestly hyperactive mutation, E451Q (8). Mutations at position 450 were introduced via site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene). All control and mutant Tnp constructs in this study contained three hyperactive mutations, E54K (19), M56A, and L372P (15), unless otherwise specified. All Tnp mutant forms were overexpressed and purified as previously described (2). In vivo analysis of W450 substitutions. The in vivo activities of the proteins with W450 substitutions were analyzed utilizing a qualitative, colorimetric papillation assay performed as previously described (19). In this assay, transposition levels were estimated by using the total number of blue papillae and mutant Tnp’s were compared to the control Tnp. Compared to the control, the W450C, W450S, and W450R mutant forms demonstrated a hyperpapillation phenotype after 48 h incubation (Fig. 2A). It is important that these mutant proteins not only displayed a net increase in total papillae but also formed papillae faster than the control Tnp (they exhibited blue papillae 8 h earlier than the control) (data not shown). When the tryptophan group was replaced with another hydrophobic, aromatic functional group through a W450F mutation, the proteins formed papillae at the same relative rate as the control and arrived at the same relative amount of total papillae formed after 48 h. These results indicate that the hydrophobic (and possibly aromatic) properties of residue W450 are deleterious for transposition in vivo. The effect of the W450C mutation was also investigated in relation to the mutation that is likely to be involved in a spatial distortion of the C-terminal -helix and that may compromise the N terminus-C terminus inhibitory interaction, L372P (10, 15). W450C mutant Tnp (lacking L372P) was shown to be hyperactive versus a control Tnp lacking both the W450C and L372P mutations (Fig. 2B). This result indicates that the mu* Corresponding author. Mailing address: The Josephine Bay Paul Center, Marine Biological Laboratory, 7 MBL St., Woods Hole, MA 02543. Phone: (508) 289-7253. Fax: (508) 457-4727. E-mail: wsreznik @wisc.edu. Published ahead of print on 14 December 2007.