Tn 552 transposase catalyzes concerted strand transfer in vitro

Abstract

The Tn552 transposase, a member of the DDE superfamily of transposase and retroviral integrase proteins, has been expressed in soluble form. The purified protein performs concerted strand transfer in vitro, efficiently pairing two preprocessed transposon ends and inserting them into target DNA. For maximum efficiency, both participating DNA ends must contain the two adjacent transposase-binding sites that are the normal constituents of the Tn552 termini. As is the case with transposition in vivo, the insertions recovered from the reaction in vitro are f lanked by repeats of a short target sequence, most frequently 6 bp. The reaction has stringent requirements for a divalent metal ion. Concerted strand transfer is most efficient with Mg 2؉ . Although it stimulates strand transfer overall, Mn 2؉ promotes uncoupled, single-ended events at the expense of concerted insertions. The simplicity and efficiency of the Tn552 transposition system make it an attractive subject for structural and biochemical studies and a potentially useful genetic tool.The transposon Tn552 is a 6.5-kb element isolated from Staphylococcus aureus responsible for resistance to ␤-lactam antibiotics (1, 2). It carries genes for three proteins likely to be involved in its transposition: a transposase (TnpA, previously called p480); a protein similar to the Mu B and Tn7 TnsC proteins (TnpB, previously called p271), which, by analogy, may enhance transposition activity and play a role in target selection; and a resolvase (3). The presence of these three genes suggests that Tn552 transposes by consecutive formation and resolution of a cointegrate between donor and target DNAs; however, this remains to be demonstrated.The Tn552 transposase belongs to the D,D(35)E superfamily of transposase proteins (3). Members of this superfamily include the integrase proteins of retroviruses and retrotransposons, the transposases of bacterial insertion sequences of the IS3 family, bacteriophage Mu A protein, and TnsB of Tn7. The defining characteristic of this family of phosphoryl-transfer proteins is a homologous catalytic core domain containing a triad of invariant acidic residues, two aspartates and a glutamate, which are essential for catalytic activity and give the superfamily its name (3, 4). Despite their homology, transposases of the D,D(35)E superfamily form a rather divergent and heterogeneous group. They range in size from the small retroviral integrases (about 290 residues) to Mu A (663 residues) and Tn7 TnsB (702 residues), and in many cases the region of homology does not appear to extend beyond the catalytic core (160-220 residues). Extensive biochemical studies of a few members of the family have provided a remarkably detailed picture of the transposition process including the substrates and proteins involved, the assembly of the transpositionally active protein-DNA complex, and the mechanism of strand transfer (5-7). However, complete understanding has been hampered by a lack of detailed structural information; despite substantial efforts, t...

The global regulator H-NS acts directly on the transpososome to promote Tn10 transposition

“…H-NS also promotes IS903 and Tn552 transposition, but it is not yet known how it acts in these systems (Swingle et al 2004). It seems unlikely that H-NS would influence IS903 or Tn552 transposition via the same mechanism as that proposed here for Tn10 transposition because there is no evidence for IHF-mediated transpososome unfolding in these systems and they lack obvious IHF-binding sites close to the terminal inverted repeats (Derbyshire and Grindley 1992;Leschziner et al 1998). …”

HIV Integrase, a Brief Overview from Chemistry to Therapeutics

“…An alternative possibility is that aggregation of purified integrase disfavors assembly of the correct complexes with two DNA ends poised for integration; such aggregation may normally be prevented by interaction with other components of the preintegration complex. Indeed, it has been shown that improving the solubility of the closely related Tn552 transposase greatly enhances the efficiency of double-end versus single-end strand transfer (43). Perhaps the strongest suggestion that additional host proteins may not be required is the finding that disrupted HIV-1 virions, which contain few host proteins, support efficient doubleend strand transfer of exogenous viral substrate DNA (44).…”

In Vitro Transposition System for Efficient Generation of Random Mutants of Campylobacter jejuni

“…This transposon exhibits features that resemble those of Mu (12), such as the coding of a single-subunit transposase and the requirement of a single accessory protein for in vivo transposition (15). However, the ends of Tn552, consisting of only 48-bp terminal inverted repeats (10,16), are much simpler than those of Mu and Tn7. These are the only sequences required for transposition, which facilitates the engineering of Tn552 for different applications.…”

“…In contrast to Tn5 (5), Tn552 displays virtually no target preference (3,6). A previous study has demonstrated that Tn552 can insert efficiently and randomly into target DNA molecules after in vitro transposition reactions (10). These properties have allowed the use of this element as a tool for nucleotide sequencing and mutagenesis (3,6).…”

“…One hundred nanograms of pBluescript SKII plasmid (Stratagene) DNA (as a target) was mixed with 100 ng (0.1 pmol) of Tn552kan-Campy or Tn552cat DNA obtained from pSB1698 or pTG426, respectively, by digestion with BglII and subsequent agarose gel purification. The transposition reaction was initiated by the addition of 30 ng of purified Histagged TnpA transposase and carried out for 1 h at 37°C in a total volume of 20 l. The transposition reactions and the purification of TnpA were carried out as previously described (6,10). The reaction mixtures were ethanol precipitated and electroporated into E. coli DH5␣, and transposon insertions were selected by plating the transformants on Luria broth plates containing either 50 g of kanamycin or 30 g of chloramphenicol per ml to select for Tn552kan-Campy or Tn552cat, respectively.…”