A dnaC mutation in Escherichia coli that affects copy number of ColE1-like plasmids and the PriA-PriB (but not Rep-PriC) pathway of chromosomal replication restart.
Escherichia coli nusG and rho mutants, which are defective in transcription termination, are killed following transformation with several ColE1-like plasmids that lack the plasmid-encoded copy-number regulator gene rom because of uncontrolled plasmid replication within the cells. In this study, a mutation [dnaC1331(A84T)] in the dnaC gene encoding the replicative helicase-loading protein was characterized as a suppressor of this plasmid-mediated lethality phenotype. The mutation also reduced the copy number of the plasmids in otherwise wild-type strains. In comparison with the isogenic dnaC(+) strain, the dnaC mutant was largely unaffected for (i) growth on rich or minimal medium, (ii) tolerance to UV irradiation, or (iii) survival in the absence of the PriA, RecA, or RecB proteins. However, it was moderately SOS-induced and was absolutely dependent on both the Rep helicase and the PriC protein for its viability. A dnaC1331(A84T) dam mutant, but not its mutH derivative, exhibited sensitivity to growth on rich medium, suggestive of a reduced capacity in the dnaC1331(A84T) strains to survive chromosomal double-strand breaks. We propose that DnaC-A84T is proficient in the assembly of replication forks for both initiation of chromosome replication (at oriC) and replication restart via the Rep-PriC pathway, but that it is specifically defective for replication restart via the PriA-PriB pathway (and consequently also for replication of the Rom(-) ColE1-like plasmids).
Harinarayanan
R
R
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.
Gowrishankar
J
J
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002876:Chromosomes, Bacterial; D004261:DNA Replication; D018076:DNA, Complementary / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000528:radiation effects; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D005804:Genes, Lethal; D009154:Mutation; D010957:Plasmids; D051716:Replication Protein A; D013014:SOS Response, Genetics; D013489:Suppression, Genetic; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D051716:Replication Protein A; C069996:PriB protein (N-terminal)