Figure 1.—

Inducing DNA DSBs specifically stimulates TnsABC+E transposition events that occur proximal to the DSB site with a preference for hotspots. Transposition was monitored with a promoter capture assay using strain JP617 derivatives on MacConkey's media (materials and methods). (A) The graph shows the frequency of transposition indicated by the number of Lac⁺ papillae after 72 hr at 30° when DSBs were induced with yhfT-3084∷Tn10 on MacConkey's media. Error bars indicate the standard error of the mean (n = 12) (data from other positions are shown in Table 3). Circles represent the E. coli chromosome. Individual insertions from separate experiments using strains expressing the TnsABC+E proteins where DSBs were induced are indicated with arrows. Arrows outside the circle indicate right-to-left insertions, arrows inside the circle indicate left-to-right insertions. The number indicates the position of the insertion on the E. coli genome in kilobases. Insertions that occurred into one of the hotspots found in this work are indicated with an asterisk (*). The gene where the Tn10-generated DSB was induced is indicated in a box (and its position in kilobase pairs). oriC and its position in kilobases are marked. Triangles indicate the position of the 10 ter sites in the chromosome; terC is indicated with its position in kilobases. The JP617 background strains were used to induce DSBs using a Tn10 insertion at one of three positions: yhfT (yhfT-3084∷Tn10 from CAG18456) (B), recD (recD-1901∷Tn10 from CAG12135) (C), or zbi (zbi-29∷Tn10 from CAG18493) (D). Breaks were induced with pZT383 (Yes breaks) or the strain had a control plasmid pJP135 (No breaks). Tn7 transposition functions were expressed from pJP123 (TnsABC+E) or a vector that does not allow transposition was used, pCW15 (TnsABC).