Tn552 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 flanked 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 Mg2+. Although it stimulates strand transfer overall, Mn2+ 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.

Figure 1

Strand transfer reactions with Tn552kan. (A) Tn552kan: the shaded boxes represent the 23-bp binding sites at the transposon ends. The asterisks indicate the position of the ³²P label. (B) Transposition reactions (3 hr) contained 270 ng of supercoiled or PstI-linearized pUC19 as target, about 50 fmol of a mixture of unlabeled and ³²P-labeled Tn552kan as the substrate, 120 ng transposase, and 10 mM MgCl₂ or MnCl₂ as indicated. Reactions run in lanes 3, 5, 10, and 12 were digested with PstI. Reaction products were resolved by agarose gel electrophoresis and detected by both ethidium bromide staining (Right) and autoradiography (Left). The predicted structures of the reaction products are shown; thin lines and thick lines represent the substrate and target DNAs, respectively. oc, open circle form of pUC19; lin, linear pUC19; ccc, covalently closed circular form of pUC19.

Figure 2

Tn552kan insertion sites in pUC19. Sequences of the flanking repeats. The positions in pUC19, shown on the right, indicate the first nucleotide of the target repeat.

Figure 3

Requirement for the transposase binding sites in transposition. (A) Structure of the 2Km2–0Km0 series of Tn552kan elements (details are identical to those in Fig. 2). (B) Transposition reactions (2 hr) contained 10 mM Mg²⁺, 225 ng of supercoiled pUC19, and about 18 fmol of ³²P-labeled substrate as indicated. Samples in even-numbered lanes were digested with PstI. Product analysis was as before.

Figure 4

Effects of glycerol and NaCl on strand transfer activity. Reactions (1 hr) contained 180 ng (Left) or 270 ng (Right) of supercoiled pUC19 as target, about 50 fmol of ³²P-labeled 2Km2 as substrate, and 10 mM Mg²⁺. Concentrations of NaCl and glycerol were as indicated. Product analysis was as before.

Figure 5

Strand transfer reactions with oligonucleotide substrates. (A) The RI and RI&II substrates. The 23-bp transposase-binding sites are indicated with horizontal square brackets. Asterisks indicate the position of the ³²P label. (B) Strand transfer reactions contained 270 ng of supercoiled pUC19 as target, about 2 pmol of a mixture of unlabeled and ³²P-labeled RI or RI&II, 120 ng of TnpA, either 133 mM or 33 mM NaCl (lanes 5–8 and 14–17), and 10 mM MgCl₂ or MnCl₂. Reaction products were resolved by agarose gel electrophoresis and detected by both ethidium bromide staining (Lower) and autoradiography (Upper).