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At least 757 records · Page 42Linked to original sources
PubMed1983

Restriction map of DNA spanning the replication terminus of the Bacillus subtilis chromosome.

The Bacillus subtilis 168 dna-1 chromosome was labelled during sporulation with [3H]thymine for five minutes immediately before termination of replication. The isolated radioactive DNA was cleaved with BamHI (or SalI) and the resulting restriction fragments separated by agarose gel electrophoresis. The individual fragments, fractionated into a series of slices cut from the gel, were then cleaved with SalI (or BamHI) and the double-digest fragments identified by electrophoresis and fluorography. All major fragments and most minor ones present in a whole double-digest were assigned to BamHI and SalI parents. Such information enabled the construction of an unambiguous restriction map of 150 X 10(3) bases of the approximately 250 X 10(3) bases of DNA labelled in the five minutes. In conjunction with published data on the order of replication of restriction fragments as termination is approached, it was clear that most (105 X 10(3) bases) of the mapped DNA was replicated by a major fork moving in one direction towards a BamHI 24.8 X 10(3) base fragment. The 45 X 10(3) bases extending to the other side of this region were labelled only slightly, and presumably was replicated by a fork that approached the other in an opposite direction until its progress was blocked or severely impeded within this region at a site, referred to as terC, sometime (less than 5 min) earlier. The regions of the map replicated in the final 2.5 and 1.0 minute by the major fork were also identified.

Bacillus subtilis↗
PubMed2004

The replication-related organization of bacterial genomes.

The replication of the chromosome is among the most essential functions of the bacterial cell and influences many other cellular mechanisms, from gene expression to cell division. Yet the way it impacts on the bacterial chromosome was not fully acknowledged until the availability of complete genomes allowed one to look upon genomes as more than bags of genes. Chromosomal replication includes a set of asymmetric mechanisms, among which are a division in a lagging and a leading strand and a gradient between early and late replicating regions. These differences are the causes of many of the organizational features observed in bacterial genomes, in terms of both gene distribution and sequence composition along the chromosome. When asymmetries or gradients increase in some genomes, e.g. due to a different composition of the DNA polymerase or to a higher growth rate, so do the corresponding biases. As some of the features of the chromosome structure seem to be under strong selection, understanding such biases is important for the understanding of chromosome organization and adaptation. Inversely, understanding chromosome organization may shed further light on questions relating to replication and cell division. Ultimately, the understanding of the interplay between these different elements will allow a better understanding of bacterial genetics and evolution.

Chromosomes, Bacterial↗
PubMed1991

Antiparallel plasmid-plasmid pairing may control P1 plasmid replication.

The copy number of the P1 plasmid replicon is stringently controlled, giving only one or two copies per newborn cell. Control is achieved by the action of the copy-control locus incA, which contains nine repeats of the 19-basepair binding site for the plasmid-encoded initiator protein RepA. A set of five similar repeats are present in the replication origin where RepA acts to trigger initiation. Using an in vitro replication system consisting of an Escherichia coli extract, the P1 origin as a template, and purified RepA protein, we show that supercoiled DNA circles containing the incA locus block origin function in trans. Shutdown becomes complete at a 1:1 ratio of origin to incA sequences. This is not due to titration of the RepA protein, as an excess of RepA can be added without restoring activity. Rather, the incA sequences appear to block the origin by direct contact in a plasmid-plasmid pairing event. When both the origin and the incA locus are present on one plasmid, trans contacts with daughter molecules appear to predominate over cis looping. The results are consistent with a model for replication control where daughter plasmids block their own replication by a pairing in which each origin is in contact with the incA locus of its partner.

Bacterial Proteins↗
PubMed2006

Genetic and biochemical characterization of the Streptococcus pneumoniae PcrA helicase and its role in plasmid rolling circle replication.

PcrA is a chromosomally encoded DNA helicase of gram-positive bacteria involved in replication of rolling circle replicating plasmids. Efficient interaction between PcrA and the plasmid-encoded replication initiator (Rep) protein is considered a requirement for the plasmid to replicate in a given host, and thus, the ability of a Rep protein to interact with heterologous PcrA helicases has been invoked as a determinant of plasmid promiscuity. We characterized transcription of the Streptococcus pneumoniae pcrA gene in its genetic context and studied the biochemical properties of its product, the PcrA(Spn) helicase. Transcription of the pneumococcal pcrA gene was directed by promoter Pa, consisting of an extended -10 box. Promoter Pa also accounted for expression of a second essential gene, radC, which was transcribed with much lower efficiency than pcrA, probably due to the presence of a terminator/attenuator sequence located between the two genes. PcrA(Spn) displayed single-stranded DNA-dependent ATPase activity. PcrA(Spn) showed 5'-->3' and 3'-->5' helicase activities and bound efficiently to partially duplex DNA containing a hairpin structure adjacent to a 6-nucleotide 5' or 3' single-stranded tail and one unpaired (flap) nucleotide in the complementary strand. PcrA(Spn) interacted specifically with RepC, the initiator of staphylococcal plasmid pT181. Although the pneumococcal helicase was able to initiate unwinding of the RepC-nicked pT181 DNA, it was much less processive in this activity than the cognate staphylococcal PcrA protein. Accordingly, PcrA(Spn) was inefficient in in vitro replication of pT181, and perhaps as a consequence, this plasmid could not be established in S. pneumoniae.

Adenosine Triphosphatases↗
PubMed1996

Helicase-contrahelicase interaction and the mechanism of termination of DNA replication.

Termination of DNA replication at a sequence-specific replication terminus is potentiated by the binding of the replication terminator protein (RTP) to the terminus sequence, causing polar arrest of the replicative helicase (contrahelicase activity). Two alternative models have been proposed to explain the mechanism of replication fork arrest. In the first model, the RTP-terminus DNA interaction simply imposes a polar barrier to helicase movement without involving any specific interaction between the helicase and the terminator proteins. The second model proposes that there is a specific interaction between the two proteins, and that the DNA-protein interaction both restricts the fork arrest to the replication terminus and determines the polarity of the process. The evidence presented in this paper strongly supports the second model.

Adenosine Triphosphatases↗
PubMed1994

Roles of ruvA, ruvC and recG gene functions in normal and DNA damage-inducible replication of the Escherichia coli chromosome.

Induction of the SOS response in Escherichia coli activates normally repressed DNA replication which is termed inducible stable DNA replication (iSDR). We previously demonstrated that initiation of iSDR requires the products of genes, such as recA, recB and recC, that are involved in the early stages of homologous recombination. By measuring the copy number increase of the origin (oriM1) region on the chromosome, we show, in this study, that initiation of iSDR is stimulated by mutations in the ruvA, ruvC and recG genes which are involved in the late stages of homologous recombination. Continuation of iSDR, on the other hand, is inhibited by these mutations. The results suggest that Holliday recombination intermediates, left on the chromosome due to abortive recombination, arrest replication fork movement. Low levels of iSDR and sfiA (sulA) gene expression were also observed in exponentially growing ruvA, ruvC and recG mutants, suggesting that the SOS response is chronically induced in these mutants. We propose that replication forks are arrested in these mutants, albeit at a low frequency, even under the normal (uninduced) conditions.

Bacterial Proteins↗
PubMed1992

Membrane-protein phosphorylation in the Bacillus subtilis cell cycle.

Sphingosine, an inhibitor of Ca(2+)-dependent protein kinases in eukaryotic cells, inhibited initiation of DNA replication in Bacillus subtilis at a concentration of 10 microM, without inhibiting elongation. The tumor promoter 12-tetradecanoyl 13-phorbol acetate, (TPA), an activator of protein kinase C in eukaryotic cells, partially counteracted the inhibition of initiation by sphingosine. Phosphorylation of polypeptides was observed in vivo at initiation of DNA replication in B. subtilis. Sphingosine, TPA, and vancomycin affected this protein phosphorylation.

Bacillus subtilis↗
PubMed1969

Chromosome replication in Salmonella typhimurium.

The replication of the Salmonella typhimurium chromosome was studied. As with E. coli 15T(-), replication was sequential. After amino acid starvation, replication proceeded from a unique and heritable region of the chromosome. 5-Bromouracil, when substituted for thymine, did not disturb the sequence of replication nor did it initiate extra replication cycles. By labeling the origin and the terminus of the chromosome with (3)H- and (14)C-thymine, respectively, it was possible to determine that the rate of chain elongation decreases as the growth rate decreases. No gap in the replication cycle could be observed.

Amino Acids↗
PubMed2001

Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces.

Linear plasmids and chromosomes of the bacterial genus Streptomyces have proteins of unknown characteristics and function linked covalently to their 5' DNA termini. We purified protein attached to the end of the pSLA2 linear plasmid of Streptomyces rochei, determined the N-terminal amino acid sequence, and used this information to clone corresponding genes from a S. rochei cosmid library. Three separate terminal protein genes (here designated as tpgR1, tpgR2, and tpgR3), which map to the S. rochei chromosome and to 100-kb and 206-kb linear plasmids contained in S. rochei, were isolated and found to encode a family of similar but distinct 21-kD proteins. Using tpgR1 to probe a genomic DNA library of Streptomyces lividans ZX7, whose linear chromosome can undergo transition to a circular form, we isolated a S. lividans chromosomal gene (tpgL) that we found specifies a protein closely related to, and functionally interchangeable with, TpgR proteins for pSLA2 maintenance in S. lividans. Mutation of tpgL precluded propagation of the pSLA2 plasmid in a linear form and also prevented propagation of S. lividans cells that contain linear, but not circular, chromosomes, indicating a specific and essential role for tpg genes in linear DNA replication. Surprisingly, Tpg proteins were observed to contain a reverse transcriptase-like domain rather than sequences in common with proteins that attach covalently to the termini of linear DNA replicons.

Amino Acid Sequence↗
PubMedDate not supplied

Initiation of DNA replication in delta cya mutants of Escherichia coli K12.

The purified DnaA protein has a high affinity for cyclic AMP (cAMP). Using equilibrium dialysis, we determined the K(A) value for cAMP as 0.819 muM(-1). The number of cAMP binding sites per DnaA protein molecule was calculated to be 1.04. This binding was quite specific for cAMP. ATP was also bound by DnaA protein and inhibited cAMP binding. This inhibition was non-competitive in nature with an inhibition constant (K(i)) of about 8.25 muM. However, in vivo we have found not only that the DnaA protein level is reduced in a cyclase deletion mutant strain, Delta++ cya, but also that DnaA protein is not degraded. The Delta cya mutants of E. coli are unable to continue DNA synthesis in the absence of de novo protein synthesis and the initiation of DNA replication in these mutants takes place from oriC.

Adenylyl Cyclases↗
PubMed2004

Replication fork and SeqA focus distributions in Escherichia coli suggest a replication hyperstructure dependent on nucleotide metabolism.

Replication from the origin of Escherichia coli has traditionally been visualized as two replisomes moving away from each other, each containing a leading and a lagging strand polymerase. Fluorescence microscopy studies of tagged polymerases or forks have, however, indicated that the polymerases may be confined to a single location (or a few locations in cells with overlapping replication cycles). Here, we have analysed the exact replication patterns of cells growing with four different growth and replication rates, and compared these with the distributions of SeqA foci. The SeqA foci represent replication forks because the SeqA protein binds to the newly formed hemimethylated DNA immediately following the forks. The results show that pairs of forks originating from the same origin stay coupled for most of the cell cycle and thus support the replication factory model. They also suggest that the factories consisting of four polymerases are, at the time immediately after initiation, organized into higher order structures consisting of eight or 12 polymerases. The organization into replication factories was lost when replication forks experienced a limitation in the supply of nucleotides or when the thymidylate synthetase gene was mutated. These results support the idea that the nucleotide synthesis apparatus co-localizes with the replisomes forming a 'hyperstructure' and further suggest that the integrity of the replication factories and hyperstructures is dependent on nucleotide metabolism.

Bacterial Outer Membrane Proteins↗
PubMed1991

The replication of plasmid pSC101.

The origin of replication of plasmid pSC101 presents features reminiscent of those found in a number of plasmids. As for those plasmids, many details about the way it initiates its replication are beginning to be known, but the regulation of this process will not be easily understood.

Bacterial Proteins↗
PubMed1974

R6K plasmid replication: influence of chromosomal genotype in minicell-producing strains of Escherichia coli K-12.

Alkaline sucrose velocity sedimentation and cesium chloride-ethidium bromide equilibrium centrifugation have been used to determine the number of copies per chromosomal equivalent of the relaxedly replicating R6K plasmid (a conjugative plasmid conferring ampicillin and streptomycin resistance) in two minicell-producing strains of Escherichia coli K-12. In one strain, the average number of covalently closed circular R6K molecules per chromosomal equivalent is 13 in log-phase and 35 in stationary-phase cells. In the other strain, there is an average of six covalently closed circular R6K molecules per chromosomal equivalent in both log- and stationary-phase cells. Selection from this strain of spontaneously occurring mutants resistant to high concentrations of ampicillin has been accomplished and such mutants show a two- to threefold increase in the number of R6K copies per chromosomal equivalent. Relative to the parental strain, mutants display the following properties: (i) elevated streptomycin resistance, (ii) a 10-fold increase in R6K conjugal transfer, (iii) a 10-fold increase in the amount of R6K plasmid deoxyribonucleic acid segregated into minicells, and (iv) a two- to threefold increase in R6K-specified beta-lactamase. The mutation(s) responsible for the increase in the number of R6K molecules per chromosomal equivalent is located on the bacterial chromosome. No R6K-linked mutations conferring the above phenotypes have been obtained. The mutations are presumed to be in chromosomal genes which play a role in the regulation of R6K replication in this strain.

Cell-Free System↗
PubMed1971

6(p-Hydroxyphenylazo)-uracil: a reversible, selective inhibitor of the replication of deoxyribonucleic acid of staphylococcal bacteriophage P11-M15.

6(p-Hydroxyphenylazo)-uracil (HPUra), a selective inhibitor of the semiconservative replication of deoxyribonucleic acid (DNA) of gram-positive bacteria, was found to inhibit the replication of DNA of bacteriophage P11-M15, a virulent derivative of the temperate Staphylococcus aureus bacteriophage P11. At appropriate concentration, HPUra inhibited DNA synthesis by P11-M15-infected S. aureus immediately and completely, regardless of the stage of the lytic cycle at which infected cells were exposed to drug. The effect of HPUra was reversible since the capacity of inhibited, infected cells to replicate phage DNA and produce mature phage could be restored by removal of HPUra from incubation media. Concentrations of HPUra which completely inhibited the replication of P11-M15 in its drug-sensitive host did not inhibit the replication of this phage or its DNA in several drug-resistant host mutants. HPUra also did not inhibit the replication of two other serologically distinct, virulent staphylococcal bacteriophages, P1 and 44AHJD, in drug-sensitive hosts.

Adenine↗