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At least 523 records · Page 29Linked to original sources
PubMed1983

Replication of Drosophila chromosomes. IX. Stimulation of initiation of polytene replication cycles in vitro by juvenile hormone.

A greater proportion of polytene nuclei show [3H]thymidine incorporation when third instar larval salivary glands of Drosophila nasuta are pulse-labelled after in vitro culture (3-24 h) in the presence of a juvenile hormone mimic, ZR 515. In glands chronically labelled with [3H]thymidine in the presence of ZR 515, more nuclei are seen to have entered new polytene replication cycles. Similarly, when salivary glands from larvae fed on 5-fluorodeoxyuridine to block polytene replication cycles at intersynthetic periods were cultured in vitro, new polytene replication cycles were initiated more quickly in the presence of ZR 515. These results suggest a stimulatory effect of juvenile hormone on new polytene replication cycles.

Animals↗
PubMed1988

Initiation of lambda DNA replication reconstituted with purified lambda and Escherichia coli replication proteins.

Using highly purified bacteriophage lambda and E. coli replication proteins, we were able to reconstitute an in vitro system capable of replication ori lambda-containing plasmid DNA. The addition of a new E. coli factor, the grpE gene product, to this replication system reduced the level of dnaK protein required for efficient DNA synthesis by at least 10-fold, and also allowed the isolation of a stable DNA replication intermediate. Based on all available information, we propose a molecular mechanism for the action of the dnaK and grpE proteins during the prepriming reaction leading to lambda DNA synthesis.

Bacterial Proteins↗
PubMedDate not supplied

Analysis of replication of DEB-alkylated DNA in yeast: bypass replication in a rad3 mutant of Saccharomyces cerevisiae.

We presented indirect evidence that in an excision-deficient rad3 mutant of yeast exposed to diepoxybutane (DEB), DNA synthesis continued past the damaged sites. This bypass replication was confined to the first post-treatment round of replication and was followed by inhibition of DNA synthesis. Analyses by alkaline sucrose gradient sedimentation and by alkaline elution from filters revealed that in mutant cells the first post-treatment round of replication proceeded at a similar rate to that in untreated cells and was not accompanied by strand scission of template DNA. The post-treatment synthesis was presumably of an error-prone type, as the frequency of reversion to ade2-1 prototrophy was increased. In contrast, in the isogenic wild-type strain, the post-treatment incorporation of radioactivity into DNA was slightly reduced and newly replicated DNA fragments were of lower molecular weight than in control cells. There was also some strain scission in template DNA, presumably resulting from excision-repair.

Alkylation↗
PubMed2004

Identification of novel initiation sites for human DNA replication around ARSH1, a previously characterized yeast replicator.

Replication of mammalian chromosomes depends on the activation of a large number of origins of DNA replication distributed along the chromosomes. We have focused our attention on a human DNA region, named ARSH1, localized to chromosome 2, that had been previously shown to act as an episomal origin in the yeast Saccharomyces cerevisiae. In the present study we have used a nascent strand DNA abundance assay to map initiation sites for DNA replication in in vivo human chromosomes around a 5 kb region encompassing ARSH1. This analysis applied to a 1-1.4 kb nascent DNA strand fraction isolated from normal skin fibroblasts revealed the presence of two major initiations sites surrounding the ARSH1 region. With an equivalent DNA fraction obtained from HeLa cells, in addition to these sites, a broad initiation profile was observed which included the ARSH1 region. This DNA region however was not sufficient to support episomal replication of an ARSH1-containing plasmid transfected into HeLa cells.

Base Sequence↗
PubMed2002

A novel strand-specific RT-PCR for detection of hepatitis C virus negative-strand RNA (replicative intermediate): evidence of absence or very low level of HCV replication in peripheral blood mononuclear cells.

Hepatitis C virus (HCV) is reported to be lymphotropic under certain circumstances. In order to evaluate viral replication in peripheral blood mononuclear cells (PBMCs), a novel strand-specific RT-PCR was developed for the determination of HCV negative-strand RNA. The detection limit of this strand-specific RT-PCR was 100 copies of HCV negative-strand RNA in the presence of 1 microg liver RNA and 10(7)-10(8) copies of positive-strand RNA. False positive PCR signals occurred only when HCV positive-strand RNA exceeded 10(9) copies. With this RT-PCR, the replicative-intermediates could be detected specifically in eight of ten liver tissues, but not in any samples of serum or plasma (0/65) of patients with chronic hepatitis C. When examining the PBMCs of 46 hepatitis C patients, including 12 patients who had undergone orthotopic liver transplantation, HCV negative-strand RNA was detected in only one patient (1/46). In addition, HCV replicative intermediates were not detected in PBMCs of patients using immunosuppressive agents. It is concluded that the replication of HCV in PBMCs is very unusual.

Hepacivirus↗
PubMed1982

Both parental deoxyribonucleic acid strands at each replication fork of replicating simian virus 40 chromosomes are cut by a single-strand-specific endonuclease.

We have measured the relative accessibility to a single-strand-specific endonuclease of the single-stranded DNA on the leading and lagging sides of replication forks in replicating simian virus 40 (SV40) chromosomes. To do this we have digested replicating SV40 chromosomes with a single-strand-specific endonuclease (P1 nuclease) and then characterized the intermediate and final products of digestion by sucrose gradient sedimentation and agarose gel electrophoresis. P1 nuclease rapidly and specifically cleaves parental DNA strands at replication forks, yielding intermediate and final cleavage products which are consistent with an approximately equal rate of nuclease cleavage on both sides of the fork. Thus, single-stranded DNA is approximately as accessible to P1 nuclease on the leading side of the fork as on the lagging side; the simplest interpretation of this observation is that the stretch of single-stranded DNA on the leading side is as long as that on the lagging side.

Centrifugation, Density Gradient↗
PubMed1997

Strand specificity in the interactions of Escherichia coli primary replicative helicase DnaB protein with a replication fork.

The interactions of the Escherichia coli primary replicative helicase DnaB protein, with synthetic DNA replication fork substrates, having either a single arm or both arms, have been studied using the thermodynamically rigorous fluorescence titration techniques. This approach allows us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal (fluorescence) and the degree of binding. Subsequently, the formation of the complexes, with different replication fork substrates, has also been characterized using the sedimentation velocity technique. To our knowledge, this is the first quantitative characterization of interactions of a hexameric helicase with replication fork substrates. In the presence of the ATP nonhydrolyzable analog, AMP-PNP, the E. coli DnaB helicase preferentially binds to the 5' arm of the single-arm fork substrate with an intrinsic affinity 6-fold higher than its affinity for the 3' arm. ATP hydrolysis is not necessary for formation of the helicase-fork complex. The asymmetric interactions are consistent with the 5' --> 3' directionality of the helicase activity of the DnaB protein and most probably reflects a preferential 5' --> 3' polarity in the helicase binding to ssDNA, with respect to the ssDNA backbone. The double-stranded part of the fork contributes little to the free energy of binding. The data indicate a rather passive role of the duplex part of the fork in the binding of the helicase. This role seems to be limited to impose steric hindrance in the formation of nonproductive complexes of the enzyme with the fork. Quantitative analysis of binding of the helicase to the two-arm fork substrate shows that two DnaB hexamers can bind to the fork, with each single hexamer associated with a single arm of the fork. In this complex, the intrinsic affinity of the DnaB hexamer for the 5' arm in a two-arm fork is not affected by the presence of the 3' arm. Moreover, the results show that the 3' arm is in a conformation which makes it easily available for the binding of the next DnaB hexamer. Because of the large size of the DnaB hexamer, the data indicate that the 3' arm is separated from the 5' arm. The separation of both arms must be to such an extent that the 3' arm can bind an additional large DnaB hexamer. These results reveal that the 3' arm is not engaged in thermodynamically stable interactions with the helicase hexamer, when it is bound in its stationary complex to the 5' arm of the fork. The significance of the these results for a mechanistic model of the hexameric DnaB helicase action is discussed.

Bacterial Proteins↗
PubMed1998

Complex of Escherichia coli primary replicative helicase DnaB protein with a replication fork: recognition and structure.

Interactions of the Escherichia coli replicative helicase DnaB protein, with DNA replication fork substrates, have been studied using rigorous fluorescence titration, fluorescence energy transfer, and analytical ultracentrifugation methods. DnaB binds the 5' single-arm fork, the 3' single-arm fork, and the two-arm fork with stoichiometries of 1, 1, and 2 DnaB hexamers per fork, independent of the length of the duplex part of the fork. Within the structurally heterogeneous binding site, the helicase accesses most of the 20 nucleotide residues of an arm. The dsDNA of the fork does not contribute to the affinity; however, it affects the positioning of the enzyme on the 5' or 3' arm. Fluorescence energy transfer experiments provide direct evidence that the DnaB helicase binds the 5' arm of the fork in a single orientation, with respect to the duplex part of the fork. The 33-kDa domains of the hexamer face the dsDNA, while the small 12-kDa domains face the 5' end of the arm. In the complex with the 3' arm, the helicase is bound in an opposite orientation when compared to the 5' arm. This is the first determination of the strict, single orientation of a helicase in the complex with a replication fork. The 3' arm accommodates a DnaB hexamer, while another hexamer is associated with the 5' arm. The complex of two DnaB hexamers bound in opposite orientations with each arm of the fork may play an important role during bidirectional replication of the E. coli DNA.

Adenylyl Imidodiphosphate↗
PubMed1996

Transcriptional activation of the dnaA gene encoding the initiator for oriC replication by IciA protein, an inhibitor of in vitro oriC replication in Escherichia coli.

Transcription of the Escherichia coli dnaA gene, encoding DnaA protein required for initiation of chromosomal DNA replication at oriC in E. coli, starts from two promoters, 1P and 2P. Gel-shift and DNase I-protection assays revealed that IciA protein, an inhibitor of initiation of in vitro E. coli chromosomal DNA replication at oriC, bound to two sites in the dnaA promoter region. One site is located upstream of promoter 1P, and the second is located downstream of promoter 2P. Whereas IciA protein did not affect transcription from the promoter 2P, transcription from the promoter 1P was specifically enhanced by IciA protein in vivo and in vitro. DnaA protein bound to the DnaA box between the two promoters 1P and 2P, acts as an transcriptional repressor. Under this condition, IciA protein counteracted the repressive effect of DnaA protein on the promoter 1P. These findings suggest that IciA protein may regulate the initiation of chromosomal DNA replication at oriC by controlling expression of the dnaA gene, as well as by inhibiting the initiation of chromosomal DNA replication at oriC.

Bacterial Proteins↗
PubMed1972

Electron microscopic studies of replicating and catenated colicin factor E1 DNA isolated from minicells (DNA replication).

Replicating and catenated DNA molecules of colicin factor E1 that were isolated from minicells were observed by electron microscopy. Two new replicating structures that have been found include: molecules that contain two untwisted replicated branches of the same length and a twisted unreplicated branch, and a twisted circular molecule with a long tail. Other molecular forms identified include circular dimers, three types of catenated dimers (open-open, open-twisted, and twisted-twisted), catenated dimers in which one of the linked molecules is replicating, catenated trimers, and a tetamer.

Centrifugation, Density Gradient↗
PubMed1972

Replication of mitochondrial DNA in mouse L cells and their thymidine kinase - derivatives: displacement replication on a covalently-closed circular template.

Thymidine kinase(-) lines of mouse L cells incorporate thymidine exclusively into mitochondrial DNA. This fact permits assessment of labeled mitochondrial DNA components in ethidium bromide-cesium chloride gradients. Contaminating nuclear DNA is unlabeled and need not be removed. Elimination of the DNase treatment of purified mitochondria reveals that the replicative forms that exhibit displacement replication up to full genome size are covalently-closed circular molecules. Denaturation followed by brief renaturation of these larger replicating molecules produces closed-circular DNA with a deficiency of Watson-Crick turns, appearing as single-strand loops. This result suggests that displacement replication proceeds with nicking and rapid closure of the covalently-closed circular template.

Animals↗
PubMed1974

Replication of colicin E1 plasmid DNA in cell extracts. II. Selective synthesis of early replicative intermediates.

The major products of colicin E1 plasmid DNA synthesis in cell extracts are completely replicated molecules and a class of molecules containing newly synthesized small DNA fragments. The addition of 10% glycerol and/or 2 mM spermidine to extracts blocks synthesis of completely replicated molecules while enhancing synthesis of molecules containing newly synthesized DNA fragments. The latter molecules, which contain on the average two DNA fragments of approximately 6 S, are early replicative intermediates for synthesis of completely replicated molecules. Synthesis of the intermediates is sensitive to rifampicin and depends on RNA synthesis. RNA components are linked to the 6S DNA molecules.

Centrifugation, Density Gradient↗
PubMed1975

Reconstruction of bacteriophage T4 DNA replication apparatus from purified components: rolling circle replication following de novo chain initiation on a single-stranded circular DNA template.

The protein products of T4 bacteriophage genes 41, 43, 45, 44, and 62 have been purified to near homogeneity using an assay which measures their stimulation of DNA synthesis in a crude lysate of Escherichia coli cells in fected by an appropriate mutant phage. When all of these proteins and T4 gene 32 protein are incubated in the presence of deoxyribonucleoside and ribonucleoside triphosphates, extensive DNA synthesis occurs on both single and double-stranded DNA templates. Analysis of this in vitro system reveals most of the features attributed to in vivo DNA replication: (1) De novo DNA chain initiation is found on a single-stranded DNA template only if ribonucleoside triphosphates are present (as expected for RNA priming of Okazaki pieces on the "lagging" strand of a replication fork). (2) With single-stranded circular DNA as template, synthesis continues for many doublings. The products after extensive synthesis resemble a rolling circle as visualized in the electron microscope, with discontinuous "lagging" strand synthesis generating a long, unbranched double-stranded tail. The fact that all six mutationally identified T4 replication gene products are required for these syntheses suggests the existence of a large multienzyme complex, constituting the T4 replication apparatus.

Chromosome Mapping↗
PubMed1982

Identification of a suppressor sequence for DNA replication in the replication origin region of the Bacillus subtilis chromosome.

The first replicating fragment of the Bacillus subtilis chromosome, B7, inhibited the replication of the plasmid that carried this fragment. In earlier work using sequential cleavage by Alu I, the suppressor function was located within a 489-base-pair segment. The nucleotide sequence of the entire segment now has been determined. The sequence is characterized by two promoter-like structures and several putative recognition sequences, such as termination signals, 2-fold symmetries, inverted repeats, and repeats. By means of sequential cleavage with exonuclease BAL-31, the essential region for suppression was located in a 200-base-pair region that contains the two promoters with the same orientation. Specific transcription was produced in vitro by using B. subtilis or Escherichia coli RNA polymerases. The transcription was mostly from the second promoter. Elimination of the -35 region of the second promoter dramatically affected both inhibitory activity and in vitro transcription, suggesting that the transcriptional activity of the second promoter is involved in the cis-inhibition of DNA replication. The significance of the suppressor sequence in the region of the replication origin of the B. subtilis chromosome is discussed.

Bacillus subtilis↗
PubMed1987

Host control of plasmid replication: requirement for the sigma factor sigma 32 in transcription of mini-F replication initiator gene.

Replication of F factor or mini-F plasmid is strongly inhibited in the rpoH (htpR) mutants of Escherichia coli deficient in the sigma factor (sigma 32) known to be required for heat shock gene expression. Transcription of the mini-F repE gene encoding a replication initiator protein (E protein) was examined by operon fusion and by direct determination of repE mRNA. The synthesis rate and the level of repE mRNA were found to increase transiently upon temperature upshift (30 degrees C to 42 degrees C) in wild-type cells but to decrease rapidly in the rpoH mutants. Thus sigma 32 appeared to be directly involved in transcription of repE whose product, E protein, in turn activates DNA replication from the mini-F ori2 region. This scheme of host-controlled plasmid replication is further supported by the analysis of transcription in vitro: RNA synthesis can be initiated from the repE promoter by a minor form of RNA polymerase containing sigma 32 but not by the major polymerase containing the normal sigma factor sigma 70. The sigma 32-mediated transcription from the repE promoter is strongly inhibited by the E protein. We conclude that transcription of the mini-F repE gene is mediated by the host transcription factor sigma 32 and is negatively controlled by its own product.

Bacterial Proteins↗
PubMed1993

Colocalization of centromeric and replicative functions on autonomously replicating sequences isolated from the yeast Yarrowia lipolytica.

Two sequences (ARS18 and ARS68) displaying autonomous replication activity were previously cloned in the yeast Yarrowia lipolytica. The smallest fragment (1-1.3 kb) required for extrachromosomal replication of a plasmid is significantly larger in Y. lipolytica than in Saccharomyces cerevisiae. Neither autonomously replicating sequence (ARS) is homologous with known ARS or centromere (CEN) consensus sequences. They share short regions of sequence similarity with each other. These ARS fragments also contain Y. lipolytica centromeres: (i) integration of marker genes at the ARS loci results in a CEN-linked segregation of the markers, (ii) an ARS on a plasmid largely maintains sister chromatid attachment in meiosis I, and (iii) integration of these sequences at the LEU2 locus leads to chromosome breakage. Deletions performed on ARS18 show that CEN and ARS functions can be physically separated, but both are needed to establish a replicating plasmid.

Base Sequence↗
PubMed1998

Role of the core DNA polymerase III subunits at the replication fork. Alpha is the only subunit required for processive replication.

The DNA polymerase III holoenzyme is composed of 10 subunits. The core of the polymerase contains the catalytic polymerase subunit, alpha, the proofreading 3'-->5' exonuclease, epsilon, and a subunit of unknown function, theta. The availability of the holoenzyme subunits in purified form has allowed us to investigate their roles at the replication fork. We show here that of the three subunits in the core polymerase, only alpha is required to form processive replication forks that move at high rates and that exhibit coupled leading- and lagging-strand synthesis in vitro. Taken together with previous data this suggests that the primary determinant of replication fork processivity is the interaction between another holoenzyme subunit, tau, and the replication fork helicase, DnaB.

Bacterial Proteins↗
PubMed2000

A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control.

We have examined a replication terminus (psiL1) located on the left arm of the chromosome of Bacillus subtilis and within the yxcC gene and at or near the left replication checkpoint that is activated under stringent conditions. The psiL1 sequence appears to bind to two dimers of the replication terminator protein (RTP) rather weakly and seems to possess overlapping core and auxiliary sites that have some sequence similarities with normal Ter sites. Surprisingly, the asymmetrical, isolated psiL1 site arrested replication forks in vivo in both orientations and independent of stringent control. In vitro, the sequence arrested DnaB helicase in both orientations, albeit more weakly than the normal Ter1 terminus. The key points of mechanistic interest that emerge from the present work are: (i) strong binding of a Ter (psiL1) sequence to RTP did not appear to be essential for fork arrest and (ii) polarity of fork arrest could not be correlated in this case with just symmetrical protein-DNA interaction at the core and auxiliary sites of psiL1. On the basis of the result it would appear that the weak RTP-L1Ter interaction cannot by itself account for fork arrest, thus suggesting a role for DnaB-RTP interaction.

Bacillus subtilis↗