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At least 739 records · Page 41Linked to original sources
PubMed1977

SP-10 bacteriophage-specific nucleic acid and enzyme synthesis in Bacillus subtilis W23.

Bacillus subtilis W23 was infected with a clear-plaque variant of SP-10 phage, namely, SP-10c. Exogenous thymidine was not incorporated into phage DNA (even in the presence of deoxyadenosine), nor was there any transfer of thymidine nucleotides from bacterial to viral DNA. The lytic program was unaffected by concentrations of 5-fluorodeoxyuridine sufficient to reduce bacterial DNA synthesis by greater than 95%. Although these data are consistent with the interpretation that thymidine nucleotides are excluded from phage DNA, formic acid digests of SP-10c DNA contained what appeared to be the four conventional bases; however, adenine and thymine were not recovered in equimolar yields. DNA-RNA hybridization and hybridization competition experiments were done. Synthesis of host RNA started to wane moments postinfection and stopped completely by 36 min. SP-10c coded for discrete classes of early and late RNA. The possibility of discrete subclasses of early RNA exists. Replication of the bacterial genome appeared to terminate 12 min postinfection. Degradation of the host DNA to acid-soluble material started at 36 min and, by the end of the latent period, greater than 90% of the host chromosome was hydrolyzed. Four apparent phage-coded enzymes have been identified. A di- and triphosphatase degraded dUTP, dUDP, dTTP, and dTDP (and, to a lesser extent, dCDP and d CTP) to the corresponding monophosphates; the enzyme had no apparent activity on dATP and dGTP. SP10c also coded for a DNA-dependent DNA polymerase, lysozyme, and a nuclease that degrades native bacterial DNA. Judging from the dependence of enzyme synthesis on the time of addition of rifampin (an inhibitor of the initiation of RNA synthesis), messengers for the di- and triphosphatase, as well as the nuclease, are transcribed from promoters that start to function 6 min postinfection. Promoters for polymerase and lysozyme did not become functional until 8 and 16 min postinfection, respectively.

Aminoglycosides↗
PubMed1997

sfi-independent filamentation in Escherichia coli Is lexA dependent and requires DNA damage for induction.

In Escherichia coli, damage to DNA induces the expression of a set of genes known collectively as the SOS response. Part of the SOS response includes genes that repair DNA damage, but another part of the response coordinates DNA replication and septation to prevent untimely cell division. The classic SOS gene product that inhibits cell division is SfiA (or SulA), which binds to FtsZ and prevents septum formation until the DNA damage has been repaired. However, another pathway acts to coordinate DNA replication and cell division when sfiA, or the sfi-dependent pathway, is inoperative. Until recently, little was known of this alternative pathway, which is called the sfi-independent pathway. We report here that sfi-independent filamentation is suppressed by lexA(Ind-) mutations, suggesting that derepression of the LexA regulon is necessary for sfi-independent induction. However, expression of LexA-controlled genes is not sufficient; DNA damage is also required to induce this secondary pathway of cell division inhibition. Furthermore, we postulate that loss of the common regulatory circuitry of the sfi-dependent and sfi-independent pathways by recA or lexA mutants uncouples cell division and DNA replication.

Bacterial Proteins↗
PubMed1999

Escherichia coli SeqA protein affects DNA topology and inhibits open complex formation at oriC.

Chromosome replication in Escherichia coli is initiated by the DnaA protein. Binding of DnaA to the origin, oriC, followed by formation of an open complex are the first steps in the initiation process. Based on in vivo studies the SeqA protein has been suggested to function negatively in the initiation of replication, possibly by inhibiting open complex formation. In vitro studies have shown that SeqA inhibits oriC-dependent replication. Here we show by KMnO(4) probing that SeqA inhibits open complex formation. The inhibition was not caused by prevention of DnaA binding to the oriC plasmids, indicating that SeqA prevented strand separation in oriC either directly, by interacting with the AT-rich region, or indirectly, by changing the topology of the oriC plasmids. SeqA was found to restrain the negative supercoils of the oriC plasmid. In comparison with the effect of HU on plasmid topology, SeqA seemed to act more cooperatively. It is likely that the inhibition of open complex formation is caused by the effect of SeqA on the topology of the plasmids. SeqA also restrained the negative supercoils of unmethylated oriC plasmids, which do not bind SeqA specifically, suggesting that the effect on topology is not dependent on binding of SeqA to a specific sequence in oriC.

Bacterial Outer Membrane Proteins↗
PubMed1972

Effect of growth conditions on the formation of the relaxation complex of supercoiled ColE1 deoxyribonucleic acid and protein in Escherichia coli.

Colicinogenic factor E1 (ColE1) is present in Escherichia coli strain JC411 (ColE1) cells to the extent of about 24 copies per cell. This number does not appear to vary in situations which give rise to twofold differences in the amount of chromosomal deoxyribonucleic acid (DNA) present per cell. If cells are grown in the absence of glucose, approximately 80% of the ColE1 molecules can be isolated as strand-specific DNA-protein relaxation complexes. When glucose is present in the medium, only about 30% of the plasmid molecules can be isolated as relaxation complexes. Medium shift experiments in which glucose was removed from the medium indicate that within 15 min after the shift the majority (>60%) of the plasmid can be isolated as relaxation complex. This rapid shift to the complexed state is accompanied by a two- to threefold increase in the rate of plasmid replication. The burst of replication and the shift to the complexed state are both inhibited by the presence of chloramphenicol. Inhibition of protein synthesis in log cultures by the addition of chloramphenicol or amino acid starvation allows ColE1 DNA to continue replicating long after chromosomal replication has ceased. Under these conditions, noncomplexed plasmid DNA accumulates while the amount of DNA that can be isolated in the complexed state remains constant at the level that existed prior to treatment. In the presence of chloramphenicol, there appears to be a random dissociation and association of ColE1 DNA and "relaxation protein" during or between rounds of replication.

Bacterial Proteins↗
PubMed1983

[Temperature-sensitive replication of plasmid pKM101].

A mutant of Escherichia coli K-12, IB10 carrying the ts10 mutation has been isolated. The mutation affects replication and inheritance of pKM101 plasmid. Incubation of the mutant under non-selective conditions of 42 degrees C resulted in the formation of R-cell population. The frequency of temperature-independent clones was 2,1 X 10(-5). The defect of pKM101 replication was shown to result in growth inhibition of host cells at a non-permissive temperature. The host growth only started after elimination of the plasmid. The mechanisms are likely to exist governing the participation of plasmid gene products in processes related to host growth. The influence of ts10 mutation on replication of other plasmids was studied. It was established that ts10 did not affect replication of R6K, RP4 and Flac+ plasmids. However, replication of R15, R205 as well as of pKM101 plasmid stopped under conditions of non-permissive temperature in IB10 mutant. Obviously, ts10 mutation results in defective replication of plasmids only belonging to the N-incompatibility group (IncPN). It is shown that R6K, RP4, Flac+ plasmids are not able to correct pKM101 replication in the mutant at 42 degrees C.

Chromosomes, Bacterial↗
PubMed1985

Mechanism of transient inhibition of DNA synthesis in ultraviolet-irradiated E. coli: inhibition is independent of recA whilst recovery requires RecA protein itself and an additional, inducible SOS function.

The mechanism of the inhibition and of the recovery of DNA synthesis in E. coli following UV-irradiation was analysed in several mutants defective in repair or in the regulation of the RecA-LexA dependent SOS response. Several lines of evidence indicated that inhibition is not an inducible function and is probably due to the direct effect of lesions in the template blocking replisome movement. Recovery of DNA synthesis after UV was largely unaffected by mutations in the uvrA, recB or umuC genes. Resumption of DNA synthesis does however require protein synthesis and the regulatory action of recA. Experiments with a recA constitutive mutant and recA 200 (temperature sensitive RecA) demonstrated that RecA protein itself is directly required but is not sufficient for recovery of DNA synthesis. We therefore propose that recovery of DNA synthesis depends upon the concerted activity of RecA and the synthesis of an inducible Irr (induced replisome reactivation) factor under RecA control. We suggest that the mechanism of recovery involves the action of Irr and RecA to promote movement of replisomes past non-instructive lesions, uncoupled from polymerisation and/or that Irr and RecA are required to promote re-initiation of a stalled replication complex downstream of a UV-lesion subsequent to such an uncoupling step.

Bacterial Proteins↗
PubMed1997

Repair analysis of promutagenic (+)-anti-BPDE DNA adduct in transcriptionally active sequences of plasmid DNA in Escherichia coli.

The extent of formation and repair of promutagenic (+)-anti-BPDE-N2-dG in transcriptionally active thymidine kinase (tk) gene insert and vector DNA fragments was assessed in the (+)-anti-BPDE treated plasmid p220-tk within the Escherichia coli hosts of varying repair potential. Polyclonal antibody (BP1), specific for (+)-anti-BPDE DNA adduct, was utilized for quantitative estimation of this bulky lesion in nanograms amounts of membrane transblotted DNA fragments. A carcinogen dose-dependent quantitative antibody binding response, due to selective recognition of the major (+)-anti-BPDE adduct, was seen with various DNA fragments separated by gel electrophoresis. The sensitivity of the immunodetection at 0.2 fmol (+)-anti-BPDE DNA adduct, allowed a linear detection in the range of modification level of 0.64 x 10(-7) to 86 x 10(-7) adducts per nucleotide in plasmid DNA. Based on this sensitivity, detection of 0.07 and 0.46 (+)-anti-BPDE DNA adducts in respective tk and vector DNA fragments was achieved upon immunoanalysis of the in vitro modified DNA. Adduct concentration dependent antibody binding was independent of size of the vector or insert fragments. Antibody binding response, to DNA modified in vivo, was dependent upon the dose of (+/-)-anti-BPDE to plasmid DNA replicating within bacterial hosts. The repair of (+)-anti-BPDE DNA adducts was determined as the loss of antibody binding sites in the specific fragments of plasmid DNA within host E. coli. About 50% of the initial DNA damage was repaired from the individual fragments during 15 min post-incubation in the repair-proficient (wild-type) E. coli cells. Complete adduct removal occurred in approx. 60 min of post-incubation period. A significant (91%) decrease in the survival of mutant (uvrA- recA-) cells was observed at 4 microM (+/-)-anti-BPDE treatment without any reduction in the colony forming units in the wild-type cells. On the contrary, no repair was seen in the excision repair-deficient (uvrA-) E. coli cells. The results indicate (1) the selectivity of the immunological method and the unique ability of the (+)-anti-BPDE specific antibodies to monitor the direct loss of this promutagenic base lesion from the in vivo modified DNA (2) the role of host excision repair pathway in efficient removal of adducts from bacterial genome determines the survival of the bacterial cells and (3) the repair of (+)-anti-BPDE DNA adducts in episomally replicating, transcriptionally active sequences occur at a rapid rate presumably due to the ease of accessibility of repair enzymes to lesions within DNA.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1988

Regulation of initiation of the chromosomal replication by DnaA-boxes in the origin region of the Bacillus subtilis chromosome.

A gene homologous to the Escherichia coli dnaA gene and two flanking 'regulatory' regions which contain nine and four DnaA-boxes respectively, are located in the replication origin region of the Bacillus subtilis chromosome. Attempts to isolate an autonomously replicating fragment from these 'regulatory' regions in order to identify oriC have been unsuccessful because the DnaA-box-containing regions strongly inhibited plasmid transformation particularly when inserted into a high-copy number plasmid pUB110. Using two plasmids differing in copy number, the two regions were subdivided into three regions, A, B and C, each containing five, four and four DnaA-boxes respectively, which differed in level of inhibition of transformation. Region C is downstream of the 'dnaA' gene and inhibits transformation in high-copy but not in low-copy number plasmids. When a part of the DnaA-boxes was deleted from the incompatible plasmids, they became transformable and produced slow-growing transformants in which the initiation frequency of chromosomal replication was selectively reduced. Fast-growing revertants were found containing the same number of plasmids as the parent but with single base changes in the DnaA-boxes. These mutations were in the most highly conserved bases of the DnaA-box sequence. This indicates that a sequence-specific interaction of the DnaA-box, probably with the B. subtilis DnaA protein is responsible for the observed incompatibility and thus appears to be involved in control of initiation frequency of the chromosomal replication.

Bacillus subtilis↗
PubMed1989

Replication properties of mini-Rts1 derivatives deleted for DnaA boxes in the replication origin.

Mini-Rts1 was found to be unable to replicate in a dnaA-null mutant. However, a mini-Rts1 derivative lacking entire tandem DnaA boxes in the replication origin retained the replication ability in a dnaA+ host although its copy number was about half that of the mini-Rts1 having complete DnaA boxes. Mini-Rts1cop1 that contains a high copy number mutation in repA was found to replicate more efficiently than mini-Rts1 of wild repA when DnaA boxes were deleted. In addition, the copy number of mini-Rts1cop1 without DnaA boxes increased 1.5-fold upon removal of incI iterons, whereas that of mini-Rts1 without DnaA boxes did not increase after the iterons were deleted. These indicate that the RepAcop1 protein can initiate the replication of mini-Rts1 efficiently even when DnaA boxes are absent from the origin of replication.

Bacterial Proteins↗
PubMed1972

Molecular weight of deoxyribonucleic acid synthesized during initiation of chromosome replication in Escherichia coli.

Alkaline sucrose gradients were used to study the molecular weight of deoxyribonucleic acid (DNA) synthesized during the initiation of chromosome replication in Escherichia coli 15 TAU-bar. The experiments were conducted to determine whether newly synthesized, replication origin DNA is attached to higher-molecular-weight parental DNA. Little of the DNA synthesized after readdition of required amino acids to cells previously deprived of the amino acids was present in DNA with a molecular weight comparable to that of the parental DNA. The newly synthesized, low-molecular-weight DNA rapidly appeared in higher-molecular-weight material, but there was an upper limit to the size of this intermediate-molecular-weight DNA. This limit was not observed when exponentially growing cells converted newly synthesized DNA to higher-molecular-weight material. The size of the intermediate-molecular-weight DNA was related to the age of the replication forks, and the size increased as the replication forks moved further from the replication origin. The results indicate that the newly synthesized replication origin DNA is not attached to parental DNA, but it is rapidly attached to the growing strands that extend from the replication fork to the replication origin, or to the other replication fork if replication is bidirectional. Experiments are reported which demonstrate that the DNA investigated was from the vicinity of the replication origin and was not plasmid DNA or DNA from random positions on the chromosome.

Amino Acids↗
PubMed1997

ATP-dependent resolution of R-loops at the ColE1 replication origin by Escherichia coli RecG protein, a Holliday junction-specific helicase.

The RecG protein of Escherichia coli is a DNA helicase that promotes branch migration of the Holliday junctions. We found that overproduction of RecG protein drastically decreased copy numbers of ColE1-type plasmids, which require R-loop formation between the template DNA and a primer RNA transcript (RNA II) for the initiation of replication. RecG efficiently inhibited in vitro ColE1 DNA synthesis in a reconstituted system containing RNA polymerase, RNase HI and DNA polymerase I. RecG promoted dissociation of RNA II from the R-loop in a manner that required ATP hydrolysis. These results suggest that overproduced RecG inhibits the initiation of replication by prematurely resolving the R-loops formed at the replication origin region of these plasmids with its unique helicase activity. The possibility that RecG regulates the initiation of a unique mode of DNA replication, oriC-independent constitutive stable DNA replication, by its activity in resolving R-loops is discussed.

Adenosine Triphosphate↗
PubMed1991

In vitro roles of Escherichia coli DnaJ and DnaK heat shock proteins in the replication of oriC plasmids.

Heat shock proteins have been shown to be involved in many cellular processes in procaryotic and eucaryotic cells. Using an in vitro DNA replication assay, we show that DNA synthesis initiated at the chromosomal origin of replication of Escherichia coli (oriC) is considerably reduced in enzyme extracts isolated from cells bearing mutations in the dnaK and dnaJ genes, which code for heat shock proteins. Furthermore, unlike DNA synthesis in wild-type extracts, residual DNA synthesis in dnaK and dnaJ extracts is thermosensitive. Although thermosensitivity can be complemented by the addition of DnaK and DnaJ proteins, restoration of near wild-type replication levels requires supplementary quantities of purified DnaA protein. This key DNA synthesis initiator protein is shown to be adsorbed to DnaK affinity columns. These results suggest that at least one of the heat shock proteins. DnaK, exerts an effect on the initiation of DNA synthesis at the level of DnaA protein activity. However, our observation of normal oriC plasmid transformation ratios and concentrations in heat shock mutants at permissive temperatures would suggest that heat shock proteins play a role in DNA replication mainly at high temperatures or under other stressful growth conditions.

Bacterial Proteins↗
PubMed1979

Host cell DNA chain initiation protein requirements for replication of bacteriophage G4 replicative-form DNA.

Bacteriophages G4ev1 and G4bs1 are simple temperature-resistant derivatives of wild-type G4 as demonstrated by restriction endonuclease analyses. The rate of replication of the duplex replicative-form DNA of these phages was normal in dnaB and dnaC mutants of the host, whereas the rate was markedly reduced in a dnaG host mutant at the restrictive temperature. We conclude that G4 duplex DNA replication requires the host cell dnaG protein, but not the dnaB and dnaC proteins. The reasons for the differences between our conclusions and those based on previously published data are documented and discussed.

Bacterial Proteins↗
PubMed1971

Regulation of chromosome replication in Bacillus subtilis: effects of amino acid starvation in strain W23.

Amino acid starvation allows limited synthesis of deoxyribonucleic acid (DNA) in Bacillus subtilis strain W23. DNA synthesis increased by about 30% after leucine starvation and by about 60% after histidine starvation. Genetic analysis on the DNA synthesized after amino acid starvation showed that all genetic markers examined have replicated, regardless of which amino acid was starved for. Initially, all markers replicated equally, but upon further replication, the thr cysB and the argA to lys regions replicated ahead of their neighboring, proximal regions. This could indicate that preferred stopping sites exist in these regions or additional sites from which replication can originate reside there. The results suggest that chromosome replication continues from those sites where it had stopped during amino acid starvation.

Amino Acids↗
PubMed2004

Bacillus anthracis and Bacillus cereus PcrA helicases can support DNA unwinding and in vitro rolling-circle replication of plasmid pT181 of Staphylococcus aureus.

Replication of rolling-circle replicating (RCR) plasmids in gram-positive bacteria requires the unwinding of initiator protein-nicked plasmid DNA by the PcrA helicase. In this report, we demonstrate that heterologous PcrA helicases from Bacillus anthracis and Bacillus cereus are capable of unwinding Staphylococcus aureus plasmid pT181 from the initiator-generated nick and promoting in vitro replication of the plasmid. These helicases also physically interact with the RepC initiator protein of pT181. The ability of PcrA helicases to unwind noncognate RCR plasmids may contribute to the broad-host-range replication and dissemination of RCR plasmids in gram-positive bacteria.

Bacillus anthracis↗
PubMed1986

Replication of plasmid RK2 in vitro by a DNA-membrane complex: evidence for initiation of replication and its coupling to transcription and translation.

The following results with an in vitro replication system utilizing a plasmid RK2 DNA-membrane complex indicate that the essential trfA-encoded replication protein of RK2 is present and active in the complex. (i) A complex extracted from a conditional replication mutant of RK2, which contains a temperature-sensitive mutation in trfA, displayed extensive DNA synthesis at the permissive temperature but little activity at the restrictive temperature. A control wild-type RK2 complex showed no inhibition of DNA synthesis at the restrictive temperature. (ii) Analysis of plasmid-encoded proteins revealed that the trfA-specified replication protein and other proteins which may be involved in the replication and maintenance of RK2 are located physically in the complex. Semiconservative plasmid DNA replication by the DNA-membrane complex was indicated by density shift experiments; DNA synthesized in the presence of a heavy-density precursor banded primarily in a heavier-density area of a neutral CsCl density gradient and consisted mostly of heavy- and light-density single-stranded DNA as determined by alkaline CsCl density gradient centrifugation. Plasmid RK2 DNA replication by the DNA-membrane complex appears to be coupled to transcription and translation as indicated by the following results: the inhibitory effects of chloramphenicol on both DNA and protein synthesis by the complex; the stimulation of replication by components normally required for protein synthesis (tRNA and all the common amino acids); the synthesis of RNA and protein by the complex; and the synthesis of specific RK2-encoded proteins.

Bacterial Proteins↗