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Analysis of protein synthesis rates after initiation of chromosome replication in Escherichia coli.

ABSTRACT: The aim of this study was to investigate whether the synthesis rates of some proteins change after the initiation of replication in Escherichia coli. An intR1 strain, in which chromosome replication is under the control of an R1 replicon integrated into an inactivated oriC, was used to synchronize chromosome replication, and the rates of protein synthesis were analyzed by two-dimensional polyacrylamide gel electrophoresis of pulse-labeled proteins. Computerized image analysis was used to search for proteins whose expression levels changed at least threefold after initiation of a single round of chromosome replication, which revealed 7 out of about 1,000 detected proteins. The various synthesis rates of three of these proteins turned out to be caused by unbalanced growth and the synthesis of one protein was suppressed in the intR1 strain. The rates of synthesis of the remaining three could be correlated only to the synchronous initiation of replication. These three proteins were analyzed by peptide mass mapping and appeared to be the products of the dps, gapA, and pyrI genes. Thus, the expression of the vast majority of proteins is not influenced by the state of chromosome replication, and a possible role of the replication-associated expression changes of the three identified proteins in the cell cycle is not clear.

SUBMITTER: Bechtloff D

PROVIDER: S-EPMC103762 | biostudies-literature | 1999 Oct

REPOSITORIES: biostudies-literature

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Analysis of protein synthesis rates after initiation of chromosome replication in Escherichia coli.

Bechtloff D D Grünenfelder B B Akerlund T T Nordström K K

Journal of bacteriology 19991001 20

The aim of this study was to investigate whether the synthesis rates of some proteins change after the initiation of replication in Escherichia coli. An intR1 strain, in which chromosome replication is under the control of an R1 replicon integrated into an inactivated oriC, was used to synchronize chromosome replication, and the rates of protein synthesis were analyzed by two-dimensional polyacrylamide gel electrophoresis of pulse-labeled proteins. Computerized image analysis was used to search ...[more]

PMID: 10515917

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Project description:Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The Escherichia coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6-DnaC6 complex that is required for loading DnaB onto single-stranded DNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6-DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6-DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6-DnaC6 complexes with excess DnaC [Allen GC, Jr., Kornberg A. Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli. J. Biol. Chem. 1991;266:22096-22101; Skarstad K, Wold S. The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. Mol. Microbiol. 1995;17:825-831]. Thus, there are two mechanisms by which an imbalance in the replicative helicase and its associated loader protein can inhibit genome duplication.

Project description:Temperature sensitivity of DNA polymerization and growth of a dnaX(Ts) mutant is suppressible at 39 to 40 degrees C by mutations in the initiator gene, dnaA. These suppressor mutations concomitantly cause initiation inhibition at 20 degrees C and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression of dnaX(Ts). One dnaA(Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro. Two models have explained dnaA(Cs,Sx) suppression of dnaX, which codes for both the tau and gamma subunits of DNA polymerase III. The initiation deficiency model assumes that reducing initiation efficiency allows survival of the dnaX(Ts) mutant at the somewhat intermediate temperature of 39 to 40 degrees C by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive. The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication. We present five lines of evidence consistent with the initiation deficiency model. First, a dnaA(Cs,Sx) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type and dnaX(Ts) strains growing at 39 and 34 degrees C. These effects were shown to result specifically from the Cs,Sx mutations, because the dnaX(Ts) mutant is not defective in initiation. Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations. Third, growing the dnaA(Cs,Sx) dnaX(Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39 degrees C, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 39 degrees C incubation reduced replication to the point that growth (and, therefore, suppression) was not possible. However, suppression was possible on glycerol medium at 38 degrees C. Fourth, the dnaX(Ts) mutation can be suppressed also by introduction of oriC mutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect. Fifth, introducing a dnaA(Cos) allele, which causes overinitiation, into the dnaX(Ts) mutant exacerbated its temperature sensitivity.

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Analysis of protein synthesis rates after initiation of chromosome replication in Escherichia coli.

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Analysis of protein synthesis rates after initiation of chromosome replication in Escherichia coli.

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