Fig. 1. (A) Schematic of Tn7 higher-order nucleoprotein complexes. (Left) The donor plasmid contains a mTn7 element (thick line) flanked by white and black rectangles representing the left (Tn7L) and right (Tn7R) transposon ends, and two PflMI restriction sites (P). (Top) Formation of a complex containing Tn7L and Tn7R (L,R) held together by TnsB represented as a white oval. (Bottom) TnsABC+D promote formation of complexes containing Tn7L, Tn7R and supercoiled (Tgt_sc) or relaxed (Tgt_n) target DNAs. The TnsC and TnsD proteins are depicted as a gray oval bound to the target DNA. (B) TnsB, TnsBC+D and TnsABC+D proteins mediate the formation of Tn7 nucleoprotein complexes. The mobilities of the DNAs, relative to the mobility of a DNA ladder (the ladder is not shown), are indicated on the right side. In lane 1, the mTn7-specific probe detects PflMI fragments containing Tn7L (3.7 kb) and Tn7R (2.2 kb). The cross-hybridization band (Xhyb) arises from binding of the mTn7-specific probe to the target DNA, which is present in excess to the donor DNA. Quantification of five independent cross-linking experiments indicated that TnsABC+D (L,R)–Tgt_sc+n complex (lane 4) incorporated 4.9 ± 2.0-fold more of the total mTn7 donor DNA than the TnsBC+D (L,R)–Tgt_sc+n complex (lane 3).

Fig. 3. The TnsABC+D (L,R)–Tgt complex is competent for transposition. (A) The products of TnsABC+D in vitro transposition. The donor plasmid (solid lines) contains a mTn7 element flanked by rectangles representing the transposon ends. The target plasmid (dashed lines) contains attTn7. Both plasmids contain an NdeI (N) restriction site, which is used to linearize the DNAs after transposition has occurred facilitating their separation by gel electrophoresis. A DSB at Tn7R or Tn7L results in DSB.R and DSB.L; an excised linear transposon (ELT) is the result of DSB at both ends; a simple insertion results from joining both transposon ends to the target DNA. DSB.R and DSB.L can be joined to the target DNA, resulting in single-end joining, i.e. DSB.R-SEJ and DSB.L-SEJ, respectively. (B) In situ transposition of the TnsABC+D (L,R)–Tgt complex in the presence of Mg²⁺. The products of the transposition reaction are labeled as described in (A).

Fig. 5. TnsB and TnsC^A225V can mediate formation of a (L,R)–Tgt complex, and this reaction depends on residues within the C-terminus of TnsB. (A) TnsB alanine mutants strongly decrease in vitro TnsABC^A225V transposition. (B) TnsB alanine mutants are defective for interaction with TnsAC^A225V- and TnsC^A225V-bound target DNA.

Fig. 7. Mutation of the C-terminus of TnsB does not significantly affect TnsC-stimulated TnsAB cleavage. The reaction products are labeled as outlined in Figure 3A.

Fig. 2. (A) The TnsABC+D (L,R)–Tgt complex contains target DNA and requires a donor DNA with two Tn7 ends. Lanes 1–7 were probed with mTn7-specific DNA, and lanes 8 and 9 were probed with target-specific DNA. In lane 5, TnsB shifts the PflMI fragment containing Tn7R, labeled Tn7R+TnsB. In lane 7, just above the backbone fragment of the ΔLR⁺ plasmid, there is a new band labeled *. The mobility of this band is consistent with a complex containing a single Tn7R fragment and attTn7 target DNA. In lane 8, the target-specific probe hybridizes to the * band, as well as to the excess nicked (T_n) and supercoiled (T_sc) target DNAs. In lane 9, the target-specific probe recognizes the TnsABC+D (L,R)–Tgt_sc complex. (B) Ca²⁺ stimulates formation of the TnsABC+D and TnsBC+D (L,R)–Tgt_sc complexes. (C) ATP is required for TnsABC+D (L,R)–Tgt_sc complex formation.

Fig. 4. Mutagenesis of the C-terminus of TnsB strongly decreases transposition and interaction with the target DNA. (A) Alignment of the amino acid sequences from the C-terminus of Tn7 TnsB with Tn5468 TnsB. (B) TnsB alanine mutants are defective for TnsABC+D transposition in vitro. The reaction products are labeled as outlined in Figure 3A. Total recombination (mTn7-specific signal – unreacted donor/mTn7-specific signal) was quantified and the TnsB aa689–690 VP→AA mutant promoted ≥2.5-fold more recombination than the other TnsB alanine mutants. (C) TnsB alanine mutants are defective for TnsABC+D and TnsBC+D (L,R)–Tgt complex formation.

Fig. 6. The TnsABC+D (L,R)–Tgt complex is more stable than the TnsBC+D (L,R)–Tgt complex. (A) Design of the thermal challenge experiment. (L,R)–Tgt complexes were assembled with the TnsBC+D and TnsABC+D proteins at 30°C, then challenged for 10 min at various temperatures as indicated below. In some reactions, a 120-fold molar excess of TnsB-binding site competitor DNA (see Materials and methods) was added at the beginning of the temperature challenge. The reactions were placed at 30°C for 10 min before cross-linking and subsequent analysis. (B) The presence of TnsA stabilizes the TnsABC+D (L,R)–Tgt complex to temperature challenge.