Replication arrests during a single round of replication of the Escherichia coli chromosome in the absence of DnaC activity.
We used a flow cytometric assay to determine the frequency of replication fork arrests during a round of chromosome replication in Escherichia coli. After synchronized initiation from oriC in a dnaC(Ts) strain, non-permissive conditions were imposed, such that active DnaC was not available during elongation. Under these conditions, about 18% of the cells failed to complete chromosome replication. The sites of replication arrests were random and occurred on either arm of the bidirectionally replicating chromosome, as stalled forks accumulated at the terminus from both directions. The forks at the terminal Ter sites disappeared in the absence of Tus protein, as the active forks could then pass through the terminus to reach the arrest site, and the unfinished rounds of replication would be completed without DnaC. In a dnaC2(Ts)rep double mutant, almost all cells failed to complete chromosome replication in the absence of DnaC activity. As inactivation of Rep helicase (the rep gene product) has been shown to cause frequent replication arrests inducing double-strand breaks (DSBs) in a replicating chromosome, DnaC activity appears to be essential for replication restart from DSBs during elongation.
Maisnier-Patin
S
S
Department of Cell and Molecular Biology, Biomedical Center, Box 596, Uppsala University, S-751 24 Uppsala, Sweden.
Nordström
K
K
Dasgupta
S
S
D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics*; D017931:DNA Primers; D004261:DNA Replication / Q000235:genetics*; D004269:DNA, Bacterial / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005838:Genotype; D016133:Polymerase Chain Reaction; D018741:Replication Origin; D013936:Thymidine / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; D013936:Thymidine