Site-Directed Mutagenesis Studies of Tn 5 Transposase Residues Involved in Synaptic Complex Formation

Abstract

Transposition (the movement of discrete segments of DNA, resulting in rearrangement of genomic DNA) initiates when transposase forms a dimeric DNA-protein synaptic complex with transposon DNA end sequences. The synaptic complex is a prerequisite for catalytic reactions that occur during the transposition process. The transposase-DNA interactions involved in the synaptic complex have been of great interest. Here we undertook a study to verify the protein-DNA interactions that lead to synapsis in the Tn5 system. Specifically, we studied (i) Arg342, Glu344, and Asn348 and (ii) Ser438, Lys439, and Ser445, which, based on the previously published cocrystal structure of Tn5 transposase bound to a precleaved transposon end sequence, make cis and trans contacts with transposon end sequence DNA, respectively. By using genetic and biochemical assays, we showed that in all cases except one, each of these residues plays an important role in synaptic complex formation, as predicted by the cocrystal structure.Transposition is the movement of a defined DNA segment, a transposon, from one location to another within the same or different DNA molecules. Tn5, which is a 5.8-kbp prokaryotic transposon, consists of two inverted terminal repeats, IS50L and IS50R, that flank the genes encoding for antibiotic resistance ( Fig. 1A) (5). IS50R carries the gene for Tn5 transposase (Tnp), the only protein catalyzing Tn5 transposition steps (13). Each IS50 element is flanked by two inverted 19-bp end sequences (ESs) (12), called the outside end (OE) and the inside end (IE), that are recognized by Tnp during the transposition process ( Fig. 1) (4). During transposition, the transposon is completely excised from donor DNA and inserted into a target DNA. This movement is carried out through a conservative "cut-and-paste" mechanism (8), which is explained and shown schematically in Fig. 2. The transposition process is mediated by three factors: Tnp, a transposon (which is bracketed by two inverted ESs), and Mg 2ϩ ions. Tnp is critical in many transposition steps, including binding to the transposon DNA ESs (24), bringing the two ESs together via formation of a protein homodimer that results in synaptic complex formation, cleaving the DNA adjacent to the ESs, and inserting the transposon into a target DNA (3,7,8,(20)(21)(22)(23).The synaptic complex is a key intermediate in Tn5 transposition. This is because all catalysis occurs in trans, with the Tnp monomer that initially bound to one ES performing the catalytic events on the ES-donor backbone (DBB) border that was initially bound by the other Tnp monomer. Throughout our studies we used a previously described hyperactive mutant (E54K, M56A, L372P) of Tnp, called EK/LP, as a control (9,11,21,29,31). The previously published cocrystal structure of EK/LP bound to the OE ES (Fig. 3) (generated from PDB ID 1MUH) predicts the existence of multiple Tnp-ES DNA contacts both in cis and in trans (9). These contacts are presumed to play key roles in stabilizing the synaptic complex and in establishing de...

DNA Looping Mediated by Site-Specific SfiI–DNA Interactions

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