TY - JOUR

T1 - Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein

AU - Skarstad, K

AU - Løbner-Olesen, A

AU - Atlung, T

AU - von Meyenburg, K

AU - Boye, E

PY - 1989/7

Y1 - 1989/7

N2 - Flow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA+ gene. When the dnaA gene was induced from either the plac or the lambda pL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit. During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells. The largest increase was observed when using the stronger promoter lambda pL compared to plac. Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions. A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced. We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication. The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions.

AB - Flow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA+ gene. When the dnaA gene was induced from either the plac or the lambda pL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit. During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells. The largest increase was observed when using the stronger promoter lambda pL compared to plac. Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions. A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced. We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication. The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions.

KW - Bacterial Proteins/biosynthesis

KW - Cell Division

KW - DNA Replication

KW - DNA, Bacterial/analysis

KW - Escherichia coli/genetics

KW - Flow Cytometry

KW - Gene Expression Regulation

KW - Isopropyl Thiogalactoside/pharmacology

KW - Lac Operon

KW - Plasmids

KW - Promoter Regions, Genetic

KW - Rifampin/pharmacology

KW - Temperature

KW - Time Factors

M3 - Journal article

C2 - 2550764

VL - 218

SP - 50

EP - 56

JO - Molecular General Genetics

JF - Molecular General Genetics

SN - 0026-8925

IS - 1

ER -