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At least 703 records · Page 39Linked to original sources
PubMed1994

Escherichia coli PriA protein is essential for inducible and constitutive stable DNA replication.

Under certain conditions, Escherichia coli cells exhibit either of two altered modes of chromosomal DNA replication. These are inducible stable DNA replication (iSDR), seen in SOS-induced cells, and constitutive stable DNA replication (cSDR), seen in rnhA mutants. Both iSDR and cSDR can continue to occur in the absence of protein synthesis. They are dependent on RecA protein, but do not require DnaA protein or the oriC site. Here we report the requirement for PriA, a protein essential for assembly of the phi X174-type primosome, for both iSDR and cSDR. In priA1(Null)::kan mutant cells, iSDR is not observed after induction by thymine starvation. Replication from one of the origins (oriM1) specific to iSDR is greatly reduced by the priA1::kan mutation. cSDR in rnhA224 mutant cells deficient in RNase HI is also completely abolished by the same priA mutation. In both cases, SDR is restored by introduction of a plasmid carrying a wild-type priA gene. Furthermore, the viability of an rnhA::cat dnaA46 strain is lost at 42 degrees C upon inactivation of the priA gene, indicating the lethal effect of priA inactivation on those cells whose viability depends on cSDR. These results demonstrate that a function of PriA protein is essential for iSDR and cSDR and suggest the involvement of the PriA-dependent phi X174-type primosome in these DnaA/oriC-independent pathways of chromosome replication. Whereas ColE1-type plasmids, known to be independent of DnaA, absolutely require PriA function for replication, DnaA-dependent plasmid replicons such as pSC101, F, R6K, Rts1 and RK2 are able to transform and to be maintained in the priA1::kan strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMedDate not supplied

Analysis of the bacterial cell cycle using strains in which chromosome replication is controlled by plasmid R1.

As an alternative approach in the study of the Escherichia coli cell cycle, we have constructed strains in which chromosome replication is under the control of various plasmid R1 derivatives, IntR1 strains. The physiological properties of such strains are described. In intR1 strains, chromosome replication can be manipulated independently of cell-cycle-related control mechanisms, and the effects on cell division can be analysed. Using this approach, we have found that the timing of replication during the cell cycle is random in intR1 strains, that overreplication of the chromosome is lethal, and that chromosome replication does not trigger cell division. Current investigations include the study of the E. coli cell cycle in the absence of the DnaA protein, the effect on cell division of a specific inhibition of the initiation of chromosome replication, and the molecular basis of uni- and bidirectional replication.

Cell Cycle↗
PubMed1980

Inititation and termination of chromosome replication in Escherichia coli subjected to amino acid starvation.

Initiation and termination of chromosome replication in an Escherichia coli auxotroph subjected to amino acid starvation were examined by following the incorporation of [3H]thymidine into the EcoRI restriction fragments of the chromosome. The pattern of incorporation observed upon restoration of the amino acid showed that starvation blocks the process of initiation prior to deoxyribonucleic acid synthesis within any significant portion of the EcoRI fragment which contains the origin of replication, oriC. In this experiment, no incorporation of [3H]thymidine into EcoRI fragments from the terminus of replication was observed, nor was it found when a dnaC initiation mutant was used to prevent incorporation at the origin which might have obscured labeling of terminus fragments. Thus amino acid starvation does not appear to block replication forks shortly before termination of replication. Attempted synchronization of replication initiation by including a period of thymine starvation subsequent to the amino acid starvation led to simultaneous incorporation of [3H]-thymidine into all EcoRI fragments within the 240-kilobase region that surrounds oriC. It is shown that the thymine starvation step allowed initiation and a variable, but limited, amount of replication to occur.

Amino Acids↗
PubMed2006

A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli.

We present evidence for a complex regulatory interplay between the initiation of DNA replication and deoxyribonucleotide synthesis. In Escherichia coli, the ATP-bound DnaA protein initiates chromosomal replication. Upon loading of the beta-clamp subunit (DnaN) of the replicase, DnaA is inactivated as its intrinsic ATPase activity is stimulated by the protein Hda. The beta-subunit acts as a matchmaker between Hda and DnaA. Chain elongation of DNA requires a sufficient supply of deoxyribonucleotides (dNTPs), which are produced by ribonucleotide reductase (RNR). We present evidence suggesting that the molecular switch from ATP-DnaA to ADP-DnaA is a critical step coordinating DNA replication with increased deoxyribonucleotide synthesis. Characterization of dnaA and dnaN mutations that result in a constitutively high expression of RNR reveal this mechanism. We propose that the nucleotide bound state of DnaA regulates the transcription of the genes encoding ribonucleotide reductase (nrdAB). Accordingly, the conversion of ATP-DnaA to ADP-DnaA after initiation and loading of the beta-subunit DnaN would allow increased nrdAB expression, and consequently, coordinated RNR synthesis and DNA replication during the cell cycle.

Adenosine Diphosphate↗
PubMed1992

Mapping of the in vivo start site for leading strand DNA synthesis in plasmid R1.

We have previously constructed Escherichia coli strains in which an R1 plasmid is integrated into the origin of chromosome replication, oriC. In such intR1 strains, oriC is inactive and initiation of chromosome replication instead takes place at the integrated R1 origin. Due to the large size of the chromosome, replication intermediates generated at the R1 origin in these strains are considerably more long-lived than those in unintegrated R1 plasmids. We have taken advantage of this and performed primer extensions on total DNA isolated from intR1 strains, and mapped the free 5' DNA ends that were generated as replication intermediates during R1 replication in vivo. The sensitivity of the mapping was considerably improved by the use of a repeated primer extension method (RPE). The free DNA ends were assumed to represent normal in vivo start sites for leading strand DNA synthesis in plasmid R1. The ends were mapped to a short region approximately 380 bp away from the R1 minimal origin, and the positions agreed well with previous in vitro mappings. The same start positions were also utilized in the absence of the DnaA protein, indicating that DnaA is not required for determination of the position at which DNA synthesis starts during initiation of replication at the R1 origin.

Bacterial Proteins↗
PubMed2005

An oriC-like distribution of GATC sites mediates full sequestration of non-origin sequences in Escherichia coli.

Sequestration of newly replicated origins is one of the mechanisms required to limit initiation of Escherichia coli chromosome replication to once per generation. Origin sequestration lasts for a considerably longer period of time than the sequestration of other newly replicated regions of the chromosome. The reason for this may be the high number of GATC sites present in the origin. Alternatively, other sequence elements in the origin region may be important for its prolonged sequestration. To distinguish between these possibilities we constructed a DNA fragment containing ten GATC sites distributed with the same spacing as the ten GATC sites in the left half of oriC, but with random sequence between the GATC sites, and inserted it at a non-sequestered chromosome location. Sequestration of this GATC-cluster lasted as long as that of oriC, or even longer. The result shows that the presence of ten GATC sites, distributed as in oriC, is sufficient to cause full sequestration, and that other sequence elements most likely do not contribute to sequestration.

Base Sequence↗
PubMed1996

The Escherichia coli Fis protein prevents initiation of DNA replication from oriC in vitro.

Fis protein participates in the normal control of chromosomal replication in Escherichia coli. However, the mechanism by which it executes its effect is largely unknown. We demonstrate an inhibitory influence of purified Fis protein on replication from oriC in vitro. Fis inhibits DNA synthesis equally well in replication systems either dependent upon or independent of RNA polymerase, even when the latter is stimulated by the presence of HU or IHF. The extent of inhibition by Fis is modulated by the concentrations of DnaA protein and RNA polymerase; the more limiting the amounts of these, the more severe the inhibition by Fis. Thus, the level of inhibition seems to depend on the ease with which the open complex can be formed. Fis-mediated inhibition of DNA replication does not depend on a functional primary Fis binding site between DnaA boxes R2 and R3 in oriC, as mutations that cause reduced binding of Fis to this site do not affect the degree of inhibition. The data presented suggest that Fis prevents formation of an initiation-proficient structure at oriC by forming an alternative, initiation-preventive complex. This indicates a negative role for Fis in the regulation of replication initiation.

Bacterial Proteins↗
PubMed1972

Chromosome replication and cell division in Escherichia coli at various temperatures of growth.

The effect of temperature on the growth rate and the pattern of chromosome replication during the division cycle of Escherichia coli B/r growing in various media was investigated. The time between divisions, the time for a round of replication (C), and the time between completion of a round and cell division (D) were threefold longer at 21 C than at 37 C. At all temperatures and in all media, D equalled one-half C, suggesting that a common mechanism controls chromosome replication and the progression of the cell toward division after completion of a round of replication.

Acetates↗
PubMed1984

Mutagenic repair in Escherichia coli. X. The umuC gene product may be required for replication past pyrimidine dimers but not for the coding error in UV-mutagenesis.

Bacteria of strain TK610 uvrA-6 his-4 umuC-36, when allowed to replicate their DNA for some hours after irradiation show induction of His+ mutations when subsequently exposed to visible light. It is suggested that base pair errors can be made opposite sites of pyrimidine dimers without involvement of umuC gene product but that the latter is required for continued replication past the dimer-mismatch region. Removal of the pyrimidine dimer by photoreversal allows replication to continue thus fixing the mismatched base as as mutation.

Bacterial Proteins↗
PubMed2000

DNA replication at high resolution.

Several decades of research have delineated the roles of many proteins central to DNA replication. Here we present a structural perspective of this work spanning the past 15 years and highlight several recent advances in the field.

Bacterial Proteins↗
PubMed1991

The FIS protein binds and bends the origin of chromosomal DNA replication, oriC, of Escherichia coli.

The FIS protein (factor for inversion stimulation) is known to stimulate site-specific recombination processes, such as the inversion of the G segment of bacteriophage Mu, by binding to specific enhancer sequences. It has also been shown to activate transcription from rRNA promoters both in vitro and in vivo. We have identified a specific binding site for FIS in the center of the origin of chromosomal DNA replication, oriC. The DNA bends upon FIS binding. Occupation of the FIS site and binding of DnaA, the initiator protein, to its adjacent binding site (R3) are mutually exclusive. A fis mutant strain can not be efficiently transformed with plasmids which carry and replicate from oriC, suggesting that FIS is required for minichromosome replication.

Bacterial Proteins↗
PubMed1997

Involvement of the ribulosephosphate epimerase gene in the dnaA and dnaR functions for initiation of chromosome replication in Escherichia coli.

Initiation of replication of the Escherichia coli chromosome is rendered temperature-sensitive by the dnaR130 mutation in the prs gene that encodes phosphoribosylpyrophosphate synthetase. The thermosensitivity of the dnaR mutant is suppressed by a spontaneous mutation in rpe, the gene encoding ribulosephosphate epimerase. Disruption of the rpe gene reverses the thermosensitive growth of the dnaR mutant. The rpe-disrupted dnaR mutant exhibits extensive DNA synthesis at low and high temperatures, as does the dnaR+ rpe disruptant and the dnaR+ rpe+ strain. The thermoresistant DNA synthesis in the rpe dnaR double mutant is dnaA dependent, because the dnaA167 mutation renders the synthesis thermosensitive. The rpe-knockout mutation abolishes the ability of the dnaA115 allele to complement the defect of the dnaA167 mutant with or without the dnaR mutation and diminishes the dnaR-complementing ability of the dnaR224 allele. These results show that the rpe product is involved in the functions of the products of both dnaA and dnaR for initiation of replication of the bacterial chromosome.

Bacterial Proteins↗
PubMed1980

DNA gyrase on the bacterial chromosome: possibility of two levels of action.

In previous studies we have shown that oxolinic acid, a specific inhibitor of the A subunit of DNA gyrase, induces DNA cleavage at 100,000-base-pair intervals on the Escherichia coli chromosome. At subsaturating drug concentrations, cleavage is induced at a fraction of these sites and DNA synthesis is partially inhibited. This partial inhibition is surprisingly rapid even when few sites have been inactivated. We now report kinetic measurements suggesting that inactivation of 100,000-base-pair gyrase sites by oxolinic acid does not inhibit DNA synthesis by simply producing barriers to replication fork movement. Slowing the rate of fork movement, thus increasing the time for a fork to reach a barrier, fails to proportionately slow inhibition of DNA synthesis. Moreover, the initial, rapid phase of inhibition is followed by a slower decline that is not accelerated by increasing the frequency of barriers by raising drug concentrations. These data, when added to the observation that additional oxolinic acid-induced cleavage occurs in replicating regions of the chromosome, suggest that gyrase may function at replication forks as well as at 100,000-base-pair intervals on the chromosome.

Chromosomes, Bacterial↗
PubMed1984

Replication origin of the Bacillus subtilis chromosome determined by hybridization of the first-replicating DNA with cloned fragments from the replication origin region of the chromosome.

The replication origin (ori) on the Bacillus subtilis genome was determined by the hybridization between the first-replicating DNA region and the cloned fragments from the ori region. The first-replicating DNA region was labeled specifically by [3H]thymidine in the presence of an inhibitor for DNA polymerase during a synchronous initiation of the chromosomal replication by germinating spores starved for thymine, and isolated by a sucrose density gradient centrifugation. Most of the labeled DNA molecules are small in size (up to 1000 bases long). The 45-kb ori region was cloned first in a lambda Charon vector and then subcloned in pBR vectors. Restriction fragments from these cloned DNAs were purified by electrophoresis in agarose gels. Only one region within the 45-kb ori region shows strong hybridization with the first-replicating DNA. Restriction fragments from this region were cloned in a phage M13 vector and separated into complementary strands. Hybridization of the labeled DNA with these cloned single-stranded fragments revealed that one site of the ori is located in each strand and they are some 2-kb apart from each other. Replication starts from these sites and proceeds inwards to pass each other.

Bacillus subtilis↗
PubMed1975

Replication of R-factor R1 in Scherichia coli K-12 at different growth rates.

The R-factor R1drd-19 mediates resistance to beta-lactam antibiotics via a beta-lactamase. A strain of Escherichia coli K-12 carrying R1drd-19 was grown at different growth rates by using different carbon sources. The specific rate of production of the R1 beta-lactamase increased linearly with the growth rate and with the gene dosage. The content of R1 deoxyribonucleic acid was estimated by alkaline sucrose gradient centrifugation and by analysis of the specific rate of beta-lactamase synthesis in nutritional shift-up experiments and was found to decrease fivefold when the growth rate was increased from 0.4 to 1.8 doublings per h. The number of R1 molecules per cell decreased from six to two in the same growth range. The presence of the plasmid affected the mean cell size significantly; at a growth rate of 0.4 doublings per h the R-+ cells were on the average 50% bigger than the R-minus cells, whereas the effect was less than 10% at a growth rate of 1.8 doublings per h. Several reports in the leterature state that the initiation mass of chromosome replication is constant. In this paper it is shown that the initiation mass of R1 replication is proportional to the growth rate. Thus, the replication of the plasmid R1 and of the chromosome are independently regulated processes. It is argued that plasmid replication is under negative control.

Bacterial Proteins↗