PMID:2167836

Citation	Moriya, S, Kato, K, Yoshikawa, H and Ogasawara, N (1990) Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells' initiation potential. EMBO J. 9:2905-10
Abstract	The dnaA gene is essential for initiation of chromosomal replication in Escherichia coli. A gene homologous with the E. coli dnaA was found in the replication origin region of the Bacillus subtilis chromosome. We have now isolated a temperature sensitive mutant of the B. subtilis dnaA by in vitro mutagenesis of the cloned gene. At a nonpermissive temperature, 49 degrees C, DNA replication stops completely after 60% increase in a rich medium, while cell mass continues to increase exponentially at 2.5 times the rate at 30 degrees C. A ratio of gene frequency between purA (origin marker) and metB (terminus marker) changes gradually from 2.7 at 30 degrees C to 1.0 in 45 min at 49 degrees C, indicating completion of the ongoing replication cycle. Upon the temperature shift down to 30 degrees C after the incubation at 49 degrees C for 60 min, DNA replication resumes without delay, and the purA/metB ratio increases rapidly to 6, i.e. consecutive initiation of more than two rounds of replication. Addition of chloramphenicol at the time of the temperature shift down did not inhibit the increase in the purA/metB ratio, while rifampicin inhibited the re-initiation completely. The mutation is a single base change from C to T in the dnaA gene resulting in an amino acid substitution from Ser to Phe in the DnaA protein. The mutation was responsible for both temperature sensitive growth and the defect in initiation of chromosomal replication. We observed a remarkable correlation between the amount of DnaA protein and the amount of initiation potential accumulated during incubation at the non-permissive temperature.
Links	PubMed PMC552006
Keywords	Bacillus subtilis/genetics; Bacillus subtilis/growth & development; Bacillus subtilis/isolation & purification; Bacterial Proteins/analysis; Bacterial Proteins/genetics; Base Sequence; Blotting, Western; Cloning, Molecular; DNA Replication; Escherichia coli/genetics; Kinetics; Molecular Sequence Data; Mutation; Sequence Homology, Nucleic Acid; Temperature
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BACSU:DNAA	involved_in	GO:0006270: DNA replication initiation	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
BACSU:DNAA		GO:0006270: DNA-dependent DNA replication initiation	ECO:0000314:		P		Fig. 6	complete CACAO 2805

References

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