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At least 685 records · Page 38Linked to original sources
PubMed1979

Expression of incompatibility by derivatives of the broad host-range inc P-1 plasmid RK2.

A segment of DNA encoding incompatibility on the inc P-1 plasmid pRK248 was identified by the analysis of deletions generated in vitro, and then cloned into several unrelated and mutually compatible plasmids. These derivatives were tested for expression of P-1 incompatibility. It was demonstrated by transformation experiments that P-1 plasmids were efficiently eliminated from an E. coli host following introduction of any one of the derivatives. However, all the derivatives were compatible with each other. The cloned segment of pRK248 DNA is itself capable of autonomous replication, without being cloned into any plasmid, if plasmid-specified gene products are provided in trans. This satellite plasmid is eliminated from the cell by the inc P-1 plasmid pRK286. The results argue against a partitioning mechanism as the basis for P-1 incompatibility but are consistent with incompatibility being the consequence of negative regulation of copy number. For the inc P-1 system, susceptibility of the plasmid to elimination, but not its ability to eliminate, requires that the P-1 replication system is active.

Chromosome Mapping↗
PubMed1995

Collapse and repair of replication forks in Escherichia coli.

Single-strand interruptions in a template DNA are likely to cause collapse of replication forks. We propose a model for the repair of collapsed replication forks in Escherichia coli by the RecBCD recombinational pathway. The model gives reasons for the preferential orientation of Chi sites in the E. coli chromosome and accounts for the hyper-rec phenotype of the strains with increased numbers of single-strand interruptions in their DNA. On the basis of the model we offer schemes for various repeat-mediated recombinational events and discuss a mechanism for quasi-conservative DNA replication explaining the recombinational repair-associated mutagenesis.

Chromosomes, Bacterial↗
PubMed1994

Negative control of plasmid DNA replication by iterons. Correlation with initiator binding affinity.

Initiation of DNA replication and negative control of initiation frequency in many bacterial plasmids are mediated by multiple binding sites (iterons) for the replicon-encoded initiator protein. Here we show that a single iteron of plasmid P1, when cloned in a multicopy vector, pUC19, can control replication of a miniP1 plasmid effectively. Using this assay system, several iterons with single point mutations were analyzed. The degree of control was directly correlated with the binding affinity of the iterons for the initiator protein in vitro. The control was also unaffected whether or not a wild type iteron was flanked with strong transcription terminators. We conclude that initiator binding is the only activity of P1 iteron sequences relevant to replication control.

Bacterial Proteins↗
PubMed2001

A broad host range replicon with different requirements for replication initiation in three bacterial species.

Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria. The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication. To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors. The purified proteins were tested for activity along with E.coli DnaB at RK2 oriV. Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein. Escherichia coli or P.putida DnaB was active with either TrfA-33 or TrfA-44, while P.aeruginosa DnaB required TrfA-44 for activation. Moreover, unlike the E.coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an ATPase accessory protein. Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.

Amino Acid Sequence↗
PubMed1979

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.

Chromosome Mapping↗
PubMed1987

Enzyme systems initiating replication at the origin of the Escherichia coli chromosome.

More than ten proteins are known to participate in replication of plasmids bearing the unique origin of the Escherichia coli chromosome (oriC). Initiation of replication of oriC plasmids has been resolved into five separable stages. An initial complex formation (Stage I) requires an oriC plasmid, dnaA protein and HU protein. In the presence of ATP at a temperature of greater than 28 degrees C, a dnaB-C protein complex interacts to form a prepriming complex (Stage II). This is followed by extensive unwinding of the template that depends on the further addition of gyrase and single-strand binding protein (SSB) (Stage III). Hydrolysis of an rNTP by dnaB protein (a helicase action) and of ATP by gyrase (a swivelling action) drives the extreme unwinding of the template. This unwound template-protein complex is the substrate for priming by primase (Stage IV) and elongation by DNA polymerase III holoenzyme (Stage V). Priming of all DNA chains is done by primase; RNA polymerase functions in template activation rather than priming. DNA polymerase III holoenzyme, composed of at least seven subunits, synthesizes the DNA chains. The alpha subunit is the polymerase, the epsilon subunit is the 3'----5' exonuclease; alpha + epsilon is the proofreading activity. Following the synthesis of new DNA chains, DNA polymerase I and ribonuclease H remove the RNA primers, polymerase I fills the gaps, and ligase seals the daughter strands (Stage VI). Replication produces plasmids identical in structure and sequence to the initial template.

Base Sequence↗
PubMed1988

Mechanism of plasmid pT181 DNA replication.

The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. This nick presumably serves as the start-site of pT181 replication by extension synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. In vitro replication of a recombinant plasmid carrying two pT181 origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading-strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two PT181 origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.

Bacterial Proteins↗
PubMed1987

Hemimethylation prevents DNA replication in E. coli.

The DNA adenine methylase of E. coli methylates adenines at GATC sequences. Strains deficient in this methylase are transformed poorly by methylated plasmids that depend on either the pBR322 or the chromosomal origins for replication. We show here that hemimethylated plasmids also transform dam- bacteria poorly but that unmethylated plasmids transform them at high frequencies. Hemimethylated daughter molecules accumulate after the transformation of dam- strains by fully methylated plasmids, suggesting that hemimethylation prevents DNA replication. We also show that plasmids purified from dam+ bacteria are hemimethylated at certain sites. These results can explain why newly formed daughter molecules are not substrates for an immediate reinitiation of DNA replication in wild-type E. coli.

Bacterial Proteins↗
PubMed2005

DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coli.

Escherichia coli DNA polymerase III holoenzyme (HE) is the main replicase responsible for replication of the bacterial chromosome. E. coli contains four additional polymerases, and it is a relevant question whether these might also contribute to chromosomal replication and its fidelity. Here, we have investigated the role of DNA polymerase II (Pol II) (polB gene product). Mismatch repair-defective strains containing the polBex1 allele--encoding a polymerase-proficient but exonucleolytically defective Pol II--displayed a mutator activity for four different chromosomal lac mutational markers. The mutator effect was dependent on the chromosomal orientation of the lacZ gene. The results indicate that Pol II plays a role in chromosomal replication and that its role is not equal in leading- versus lagging-strand replication. In particular, the role of Pol II appeared larger in the lagging strand. When combined with dnaQ or dnaE mutator alleles, polBex1 showed strong, near multiplicative effects. The results fit a model in which Pol II acts as proofreader for HE-produced misinsertion errors. A second role of Pol II is to protect mismatched 3' termini against the mutagenic action of polymerase IV (dinB product). Overall, Pol II may be considered a main player in the polymerase trafficking at the replication fork.

Alleles↗
PubMed1977

Replication of plasmid pSC101 in Escherichia coli K12: requirement for dnaA function.

Replication of pSC101 was analyzed by using DNA-DNA hybridization and alkaline sucrose gradient centrifugation. Mutants of the dnaA gene were tested for their capacity to replicate pSC101 DNA at a non-permissive temperature. Only a small amount of radioactive precursor was incorporated into pSC101 DNA in dnaA mutants at 42 degrees C whereas active incorporation into plasmid DNA took place in dnaA+ strain under the same conditions. The effect of the dnaA mutation was grater on plasmid DNA synthesis than on host chromosomal DNA synthesis. The numbers of copies of pSC101 per chromosome in wild type and dnaA strains, grown at 30 degrees C, were about 8 and 2, respectively. These results indicate that the dna A gene product is required for the replication of pSC101 DNA.

Chromosomes, Bacterial↗
PubMed1994

The mutant DnaAcos protein which overinitiates replication of the Escherichia coli chromosome is inert to negative regulation for initiation.

Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30 degrees C. DnaAcos protein was purified from an overproducing strain and found to be as active as wild-type DnaA protein in initial synthesis rates of minichromosome replication in vitro at 30 degrees C. However, whereas efficient initiation occurred for only 20 min with wild-type DnaA protein, it continued for 45 min with DnaAcos protein, an indication that DnaAcos protein retained initiation activity for a longer time than wild-type DnaA protein. Also, whereas wild-type DnaA protein is inactivated by ADP binding, DnaAcos protein failed to be inactivated by ADP due to its inability to bind nucleotide. Thus, DnaAcos protein appears to lack negative regulation for its initiation activity. At 42 degrees C, a temperature at which initiation of chromosome replication is normal in the dnaAcos mutant, in vitro DnaAcos protein activity decreased to 25% of that observed at 30 degrees C. This coincident occurrence of normal initiation in vivo and reduced activity is consistent with the idea that negative control of DnaA protein activity is necessary for normal replication.

Adenosine Diphosphate↗
PubMed1998

RuvAB acts at arrested replication forks.

Replication arrest leads to the occurrence of DNA double-stranded breaks (DSB). We studied the mechanism of DSB formation by direct measure of the amount of in vivo linear DNA in Escherichia coli cells that lack the RecBCD recombination complex and by genetic means. The RuvABC proteins, which catalyze migration and cleavage of Holliday junctions, are responsible for the occurrence of DSBs at arrested replication forks. In cells proficient for RecBC, RuvAB is uncoupled from RuvC and DSBs may be prevented. This may be explained if a Holliday junction forms upon replication fork arrest, by annealing of the two nascent strands. RecBCD may act on the double-stranded tail prior to the cleavage of the RuvAB-bound junction by RuvC to rescue the blocked replication fork without breakage.

Bacterial Proteins↗
PubMed1972

Effect of thymine limitation of chromosomal deoxyribonucleic acid synthesis in Proteus mirabilis.

The effects of thymine limitation on the rates of growth, deoxyribonucleic acid (DNA) synthesis, and increase in viable cell number for a thymine auxotroph of Proteus mirabilis were investigated. At thymine concentrations of 1.0 mug/ml and below, these rates were markedly decreased. After a reduction in thymine concentration from 10 mug/ml to 0.2 mug/ml, mass synthesis continued at the preshift rate for several hours. In contrast, the rate of DNA synthesis immediately decreased, resulting in a decrease in the DNA to mass ratio to about one-half of its normal level. Viable counts remained constant for several hours after the reduction in thymine concentration, and enlarged cells and multicellular "snakes" were formed. The rate of DNA synthesis was reduced at thymine concentrations below approximately 1.7 mug/ml. The addition of thymine to cultures which had been completely starved for thymine increased the rate of DNA synthesis to at least twice its normal value; this suggests that extra rounds of chromosome replication can be induced in P. mirabilis as previously observed in Escherichia coli.

Chromosomes, Bacterial↗
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↗