Fig. 1. Repair of Mu insertions is prevented if chromosome replication is prevented.

(A) Known steps in the non-replicative (repair) pathway of Mu transposition. This pathway is used during integration of infecting Mu. The infecting genome is linear, and attached at both ends to long flanking DNA (FD) protected by Mu N protein. This DNA is variable in length (60 – 150 bp at the L end and 0.5 – 3 Kbp at the R end). MuN circularizes the DNA non-covalently, and protects it from nucleases. MuA catalyzes cleavage and strand transfer (integration) of Mu into the E. coli genome, assisted by MuB protein and host HU protein. The N protein is removed only after integration by an unknown mechanism assisted by the transpososome (purple ball), and the FD is degraded by RecBCD. Degradation is slowed in the absence of ClpX. In vitro, the final product of RecBCD degradation leaves 4 nt of the FD. This strand transfer intermediate with short flanks is likely the substrate for the final steps in repair, where the 5 bp target gaps are filled to generate a simple insertion. (B) Mu life cycle. After infection, Mu integrates into the E. coli genome, the FD is degraded, the Mu insertion is repaired, and Mu enters the lytic cycle. The approximate time (0–60 min) of these events is indicated. (C) Schematic of known mechanisms for replication initiation at oriC and during Restart of Mu replication in E. coli. (D) Preparation for the FD detection assay. At various times after infection, genomic DNA was subjected to pulse-field agarose gel electrophoresis (PFGE) to separate integrated Mu from free Mu, and the gDNA was excised for analysis by PCR. The (−) lane is an uninfected control where Mu DNA was added to the gDNA prior to electrophoresis, to assess contamination of the excised gDNA band with free Mu. Mu-length DNA at 60 min reflects packaged virions. (E) PCR assay for FD detection. Strains were infected with Mu at 37°C for the indicated times, and the isolated gDNA was tested by PCR to detect Mu or FD (lacZ) sequences using appropriate primers; lacZ sequences linked to infecting Mu are not found in the host (Au et al., 2006). + is Mu virion DNA and − is gDNA from the uninfected control lane in panel D. WT (BW25113); RecB⁻ (JW2788); ClpX⁻ (JW0428). (F) Monitoring inactivation of replication in DnaAts (SS1424) and DnaCts (SS1021) mutants by measuring chromosome equivalents. The mutants were held at 42°C for indicated times, without shaking, and fixed in ethanol before staining with the fluorescent DNA stain SYTOX Green. Stained cells were analyzed by flow cytometry as described under Experimental Procedures. By 60 min, there are no new rounds of replication in either mutant, as judged by the shift in the initial DNA content of ~2–4 chromosome equivalents to 1 chromosome equivalent. (G) FD removal depends on chromosome replication. DnaAts and DnaCts mutants were infected with Mu at both 30°C and 42°C. The latter infections were carried out after replication arrest for 90 min. In the WT (MG1655) infection at 42°C, FD is removed faster than at the lower temperatures. The PriA mutant (SS1448) and its WT parent (SS996) were infected at 30°C. Strains are listed and described in Table S1.