Target capture during Mos1 transposition

Abstract

DNA transposition contributes to genomic plasticity. Target capture is a key step in the transposition process, because it contributes to the selection of new insertion sites. Nothing or little is known about how eukaryotic mariner DNA transposons trigger this step. In the case of Mos1, biochemistry and crystallography have deciphered several inverted terminal repeat-transposase complexes that are intermediates during transposition. However, the target capture complex is still unknown. Here, we show that the preintegration complex (i.e., the excised transposon) is the only complex able to capture a target DNA. Mos1 transposase does not support target commitment, which has been proposed to explain Mos1 random genomic integrations within host genomes. We demonstrate that the TA dinucleotide used as the target is crucial both to target recognition and in the chemistry of the strand transfer reaction. Bent DNA molecules are better targets for the capture when the target DNA is nicked two nucleotides apart from the TA. They improve strand transfer when the target DNA contains a mismatch near the TA dinucleotide.

Keywords: DNA-Protein Interaction; Enzyme Mechanisms; Gene Transposable Elements; Genome Structure; Genomic Instability; Target Capture; Transposition.

FIGURE 1.

Organization of the Mos1 target capture complex. A, implemented protocol for TCC assembly. PIC was assembled with Tpase (gray ovals) and unlabeled PC-ITR (arrows) in catalytic conditions After assembly, labeled target (T*) was added with 5 mm EDTA to avoid strand transfer. TCCs were analyzed by EMSA. B, target capture assay. PIC assembly was monitored using labeled PC-ITRs (*) (lane 1). TCC assembly was monitored as described for A with labeled target (*) and unlabeled ITR (c) in the presence of transposase (+) (lane 5) and with competitor DNA (lane 6). The direct binding of MOS1 to the target was analyzed in the presence (+) or absence (−) of competitor DNA (lanes 3 and 4). Left side, complexes with labeled PC-ITR (SEC2, PIC). Right side, complexes with labeled targets (TCC and nonspecific binding complex (NSC)). Free DNAs (target, dimer of target (dtarget), and ITR) are indicated. C, PIC assembly was monitored using labeled PC-ITRs. TCC were performed with two labeled target: a short (S*) (30 bp) and a long target (L*) (50 bp), and analyzed by EMSA. D, determination of the number of target in the TCC. EMSAs were performed with short/long target combinations and a cold PC-ITR, as indicated. L, long target; S, short target. Labeled targets are indicated by asterisks. Targets present in the complexes are drawn on the right. E, determination of the number of transposase in TCC. For each condition, TCCs were assembled as described in A with labeled target (*), unlabeled ITR (c), and transposase. TCC assembly was performed without factor Xa treatment (0 h). After TCC assembly, TCCs were subjected to factor Xa cleavage for various times (1 h, 2 h 30 min, and 5 h) before EMSA. The proteins present in the various TCCs are drawn on the right. F, determination of the number of ITRs in the TCC. TCC assembly was performed with unlabeled (c) short (S)/long (L) ITR combinations and a labeled target (*) and analyzed by EMSA. The ITRs present in the complexes are drawn on the right. L/L, two long ITRs; S/L, one short and one long ITR; S/S, two short ITRs. G, kinetics of TCC formation. After PIC assembly as described in A, TCC formation was allowed to proceed for various times (0, 5, 30, 60, 120, and 180 min). The percentage of TCC formed was quantified (labeled target in TCC-labeled target in TCC + free target) and plotted as a function of time.

FIGURE 2.

TCC assembly according to the ITR ends. A, diagram of the different types of ITR used. UC-ITR, PN-ITR, and nontransferred strand (NTS) cleaved 3 bp inside the ITR; PC-ITR, nontransferred strand, and transferred strand (TS) are cleaved. B, TCC assembly with various ITRs. For each ITR, PIC assembly was assayed using labeled ITRs (*) and Mg²⁺, at either 4 or 30 °C (as specified). TCC were assembled using cold PICs and a labeled target (*) without EDTA, at 4 °C when the PICs were preformed at 4 °C (lanes 3, 7, and 11) and with or without EDTA at 30 °C when the PICs were preformed at 30 °C (lanes 4, 8, and 12). C, for quantification, the percentage of PIC was obtained by dividing the amount of PIC by the amount of PIC + free ITR. The percentage of TCC was obtained by dividing the amount of TCC by the amount of TCC + free target. The ratio TCC/PIC was calculated for each ITR and plotted on a graph, and the values are indicated in the table.

FIGURE 3.

Target commitment. A, diagram of the two-step assay used to measure target commitment. Two labeled targets different in size (short (S*) or long (L*)) were used. PIC was first assembled with unlabeled PC-ITR. During this first step, TCC was allowed to proceed with the labeled target 1 (short (S*) or long (L*)). At step 2 of the assay, a second target of distinguishable size was added. After a further incubation for 1 h at 30 °C, the complexes obtained were analyzed by EMSA. To find out whether either of the two targets is preferred for TCC formation, a mix of both targets was incubated during a 2-h assay. The same molar concentration of targets was used in each assay. B, TCC formation was performed with short (S), long (L), or a mix of short and long targets (SL) in the same molar concentrations as controls. Target commitment was assayed as described in A (lanes 2–4). Experiments were repeated six times and quantified. The amount of complex formed with the first target is divided by the amount of the complex formed with the same target when both targets are added together. The averages of target commitment are indicated below the panel. The significance of the differences (lane 2 versus lane 3 and lane 4 versus lane 3) was assayed using a Kruskal-Wallis test and resulted in a p value of 0.7.

FIGURE 4.

Integration assays. A, diagram showing the expected integration of the PC-ITR into the labeled target. The positions of the PCR primers used to amplify the integration products are indicated by arrows. B, TCCs were allowed to proceed as described for Fig. 1A, under various conditions: 4 °C with Mg²⁺ (lane 1), 30 °C with Mg²⁺ (lane 2), and 30 °C with EDTA (lane 3). Integration products were recovered and loaded onto a denaturing gel. A G+A ladder was used to calculate the sizes of the integration products, which are indicated on the right. C, three products were sequenced after PCR amplification. They all contained the ITR (bold type) integrated in the target DNA (italic type) at the TA dinucleotide (oversized uppercase type), as expected.

FIGURE 5.

Role of the TA dinucleotide at the insertion site. A, TCC assembly was done as indicated in Fig. 1A using a TA target or a GC target (which contains no TA dinucleotide) and analyzed by EMSA. The analysis was repeated five times, and the percentage of TCC for each target was obtained by dividing the amount of TCC by the amount of TCC + free target. The normalized results were plotted on a graph. B, integration assays were performed as described for Fig. 4, using cold PC-ITR and a labeled TA target or a labeled GC target. Integration products were analyzed onto denaturing gel.

FIGURE 6.

Checking the role of Arg¹⁸⁶ in target capture. PIC assembly was controlled using a labeled PC-ITR for each transposase (WT and R186A) as indicated. TCCs were assembled as described for Fig. 1.

FIGURE 7.

Target capture assays using modified targets. A, the sequence of the wild type TA target (bold type) and targets with one mismatch (M+1, M+2, and M+3) or one nick (N+1, N+2, and N+3) are given. The mismatches are boxed, and arrows indicate the nicks. B, TCCs were assayed under standard conditions (Fig. 1A), using either mismatched targets (left panel) or nicked targets (right panel). The wild type TA target (WT) is used as a reference. The resulting complexes were analyzed in EMSA. C, experiments were repeated at least three times. For each target, the relative percentage of TCC was calculated and plotted as histogram bars. The significance of the differences between the different modified targets and the TA target was assayed using a Kruskal-Wallis test. Significant p values are indicated. ***, p < 0.001; *, p < 0.05. D, integration assays were performed as in Fig. 4, using either mismatched targets (left panel) or nicked targets (right panel). The results for the nicked targets were obtained using targets labeled on both strands and compared with a similarly labeled WT target. E, experiments were repeated at least three times, quantified, and plotted as histogram bars. The significance of the differences between each of the modified targets and the WT target was assayed using a Kruskal-Wallis test. Significant p values are indicated. ***, p < 0.001. F, the percentages of TCC and integration obtained in C and E, respectively, were reported for each target. To assess the strand transfer efficiency, regardless of the target capture efficiency, the integration was normalized according to the percentage of TCC formed with each target (integration percentages divided by TCC percentages).