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PubMed2000

Chemistry and biological activity of new 3-benzazepines.

This review summarizes our experiments investigating structure-activity relationships of 3-benzazepines. Three 7, 8-dihydroxy-3-benzazepines [7-9] were cytotoxic to human promyelotic leukaemia HL-60 cells. Compound [9] showed the highest cytotoxicity and the activity was twice as high as that of dopamine (DA, [11]). Three active compounds [7-9] produced radicals, whereas other less potent benzazepines [1-6, 10] did not produce radicals. Furthermore, cytotoxic 3-benzazepines [7-9] also enhanced the decay of ascorbic acid in rat brain homogenate. Two 7,8-dimethoxy-3-benzazepines [5, 10] were able to form a complex with the replicative form of plasmid DNA. The multidrug resistance (MDR) P-glycoprotein (Pgp) efflux pump of mouse lymphoma cells was inhibited by three compounds [5, 8, 10]. Compound [8] has the highest activity in MDR reversal and is two times more potent than verapamil. Three cytotoxic 3-benzazepines [7-9] showed inhibitory effects against reverse transcriptase (RT) of Moloney leukemia.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1986

Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

A system in which transfected plasmid DNA replicates in the cytoplasm of poxvirus-infected cells is described. A variety of recombinant plasmids was introduced into poxvirus-infected cells by transfection, and replication of input plasmid DNA was monitored by (i) digestion with restriction enzymes that discriminate between input methylated plasmid DNA and unmethylated DNA produced by replication in mammalian cells; (ii) amplification of intracellular plasmid DNA; and (iii) density shift analysis in the presence of BrdUrd. Replication of plasmid DNA was observed in the cytoplasm of cells infected with the tumorigenic leporipoxviruses Shope fibroma virus (SFV) and myxoma, and less extensively with the orthopoxvirus vaccinia, but not in uninfected cells. Unexpectedly, all input plasmids tested, including pBR322, pUC13, polyoma, PM2 phi X174 replicative form (RF), and M13 RF, replicated with equal efficiency in SFV-infected cells, indicating that no specific replication origin sequence is required. The transfected plasmid DNA was replicated concomitantly with the infecting poxviral DNA and by 24 hr post-transfection, it resided predominantly in high molecular weight Dpn I-resistant head-to-tail tandem repeats. The failure to detect unreplicated Dpn I-sensitive plasmid concatemers early in replication together with the absence of significant levels of integrated plasmid sequences in the poxviral genome suggest that replication of the transfected plasmid DNA is not the consequence of nonhomologous recombination of concatemeric plasmid DNA into the poxvirus genome, but rather of an autonomous process that is dependent on trans-acting replication factors produced during virus infection, and that does not require a specific origin sequence on the substrate plasmid DNA.

Animals↗

PubMed1999

Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences.

The SeqA protein binds to the post-replicative forms of the origins of replication of the Escherichia coli chromosome (oriC) and the P1 plasmid (P1oriR) at hemimethylated GATC adenine methylation sites. It appears to regulate replication by preventing premature reinitiation. However, SeqA binding is not exclusive to replication origins: different fragments with hemimethylated GATC sites can bind SeqA in vitro when certain rules apply. Most notably, more than one such site must be present on a bound fragment. The protein appears to recognize individual hemimethylated sites, but must undergo an obligate cooperative interaction with a nearby bound protein for stable binding. SeqA contacts both DNA strands in a discrete patch at each hemimethylated GATC sequence. All four GATC bases are contacted and are essential for binding. Although the recognized sequence is symmetrical, the footprint on the methylated strand is always broader, suggesting that the bound protein is positioned asymmetrically with its orientation dictated by the position of the unique methyl group. Studies of alternative spacings and relative orientations of adjacent sites suggest that each site may be recognized by a symmetrical dimer with an induced asymmetry in one of the subunits similar to that seen with certain type II restriction endonucleases.

Bacterial Outer Membrane Proteins↗

PubMedDate not supplied

[Classical and non-classical DNA conformations].

All possible right and left double helical structures which may exist in short fragments as well in polymeric DNA have been obtained on the basis of a developed rigorous and accurate method of conformational analysis of DNA. In polymeric DNA only right regular double helices are possible with preference of B-form that is the main biological form of DNA. In contrast, for short fragments the left and right helices have practically the same energies providing some physical ground for side-by-side form, which biologically is possible as a recombination form and maybe as a replication form.

DNA↗

PubMed1972

Cis-limited action of the gene-A product of bacteriophage phiX174 and the essential bacterial site (E. coli-electron microscopy-cis-acting protein-specifically-nicked RF).

Parental replicative-form (RF(*)) DNA of bacteriophage varphiX174 in a replication-deficient host cell (rep(3) (-)) exhibits two characteristic features that correlate the function of viral gene A with the initiation of viral DNA replication: a specific discontinuity in the viral strand of a constant number of RF molecules and elongation of the viral strand to yield replicative-intermediate DNA forms with single-stranded tails. At high multiplicities of infection, these initiation events are limited to an average of four specifically nicked RFII molecules per cell. The limiting factor from the host cell may be related (or identical) to the essential bacterial sites known to limit the participation of parental genomes in RF replication. Double-infection experiments with wild-type phage and phage carrying an amber mutation in gene A show that the formation of gene A-specific RFII and RI is cis-limited to only the wild-type DNA. These results provide a basis at the DNA level for the known asymmetric complementation of gene A.

Binding Sites↗

PubMed1986

Synthesis of viral DNA forms in Nicotiana plumbaginifolia protoplasts inoculated with cassava latent virus (CLV); evidence for the independent replication of one component of the CLV genome.

Totipotent leaf mesophyll protoplasts of Nicotiana plumbaginifolia, Viviani were inoculated with cassava latent virus (CLV) or with full length copies of CLV genomic DNAs 1 and 2 excised from replicative forms of M13 clones. Virus specific DNAs began to appear 48-72h after inoculation with virus or cloned DNAs, coincident with the onset of host cell division. Infected cells accumulated supercoiled forms of DNAs 1 and 2 as well as progeny single-stranded (ss) virion (+) sense DNAs representing each component of the genome. Both supercoiled and ss molecules were synthesised by cells inoculated with cloned DNA 1 alone but DNA 2 failed to replicate independently.

Cell Division↗

PubMed2007

Transcription factor Runx1 recruits the polyomavirus replication origin to replication factories.

Eukaryotic DNA replication takes place in the replication factories, where replication proteins are properly assembled to form replication forks. Thus, recruitment of DNA replication origins to the replication factories must be the key step for the regulation of DNA replication. The transcription factor Runx1 associates with the nuclear matrix, the putative substructure of DNA replication factories. An earlier report from our laboratory showed that Runx1 activates polyomavirus DNA replication, and that this requires its nuclear matrix-binding activity. Here, we show that Runx1 activates polyomavirus DNA replication by stimulating the binding of the viral-encoded replication initiator/helicase, large T antigen, to its replication origin. We found that newly replicated polyomavirus DNA is associated with the nuclear matrix and that large T antigen is targeted to replication factories, suggesting that polyomavirus is replicated in replication factories on the nuclear matrix. Although Runx1 did not co-localize with large T antigen-containing foci by itself, it co-localized with large T antigen-containing replication factories during Runx1-dependent polyomavirus DNA replication. These observations together suggest that Runx1 recruits the polyomavirus replication origin to the replication factory on the nuclear matrix, and that this requires the nuclear matrix-binding activity of Runx1.

Animals↗

PubMed1984

Initiation of SV40 DNA replication after microinjection into Xenopus eggs.

We have examined the capacity of Xenopus laevis eggs to support replication of microinjected SV40 DNA. As previously reported, microinjected DNA undergoes semi-conservative replication. Unlabeled SV40 DNA was microinjected with [3H]dTTP and, after a 3 h incubation period, the DNA was recovered and adsorbed to BND-cellulose. Elution with an NaCl gradient removes molecules that are entirely double-stranded but not those with single-stranded regions. The latter DNA population is eluted with caffeine. The radioactive DNA that eluted with NaCl was comprised mostly of supercoiled and open circular SV40 DNAs. The radioactive DNA eluted with caffeine was comprised mainly of endogenous DNA but also contained replicative forms of SV40 DNA. Analysis of SV40 DNA replication intermediates by electron microscopy revealed mainly Cairn's forms of varying degrees of maturation. Digestion with BamH1, which cleaves SV40 DNA almost opposite the normal viral replication origin, indicated that SV40 DNA microinjected into frog eggs does not initiate DNA synthesis at its normal initiation site nor at any other obvious preferred site. Rather, it appears that when this template is injected into activated Xenopus eggs, replication may initiate at random.

Animals↗

PubMed2001

The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus.

In addition to the RI (replicative intermediate RNA) and native RF (replicative form RNA), mouse hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt solutions, approximately 70% of the replicating and transcribing structures that accumulated in infected cells by 5-6 h post-infection were multi-stranded intermediates, the RI/TIs. The other 30% were in double-stranded structures, the native RF/TFs. These replicating and transcribing structures were separated by velocity sedimentation on sucrose gradients or by gel filtration chromatography on Sepharose 2B and Sephacryl S-1000, and migrated on agarose gels during electrophoresis, according to their size. Digestion with RNase T1 at 1-10 units/microgram RNA resolved RI/TIs into RF/TF cores and left native RF/TFs intact, whereas RNase A at concentrations of 0.02 microgram/microgram RNA or higher degraded both native RF/TFs and RI/TIs. Viral RI/TIs and native RF/TFs bound to magnetic beads containing oligo(dT)(25), suggesting that the poly(A) sequence on the 3' end of the positive strands was longer than any poly(U) on the negative strands. Kinetics of incorporation of [(3)H]uridine showed that both the RI and TIs were transcriptionally active and the labelling of RI/TIs was not the dead-end product of aberrant negative-strand synthesis. Failure originally to find TIs and TF cores was probably due to overdigestion with RNase A.

Animals↗

PubMed1969

Phi-X-174 bacteriophage structural mutants which affect deoxyribonucleic acid synthesis.

Seven cistrons in phiX-174 were identified and one in particular was studied intensively: cistron A, which is assigned a protein in the mature phage. Amber mutants in this cistron synthesize a new deoxyribonucleic acid (DNA) form in addition to circular phage DNA upon infection of the restrictive host. This DNA is linear, non-infectious, and single-stranded; it is formed from the phage strand of replicative form phiX-174 DNA. These mutants produce two different defective particles in the restrictive host. One particle contains circular phage DNA but is not infectious; the other contains the new DNA form and is similar to the 70S particles found in wild-type phage lysates. The mutant A gene product acts independently of normal A protein upon mixed infection of the restrictive host with an A mutant and a mutant from any other cistron or wild type.

Bacteriophages↗

PubMed1975

The deoxyribonucleic acid unwinding protein of Escherichia coli. Properties and functions in replication.

The DNA unwinding protein of Escherichia coli (Sigal, N., Delius, H., Kornberg, T., Gefter, M., and Alberts, B. (1972) Proc. Nat. Acad. Sci. U.S.A. 69, 3537-3541) has been purified to homogeneity by a simple procedure which utilizes its stability to heating. The protein is an asymmetric tetramer of 18,500 dalton subunits which binds preferentially to single-stranded DNA at a ratio of one protein molecule per 32 nucleotides. Binding to DNA is complete in less than 10 s at 0 degrees while release of the protein from single-stranded DNA is relatively slow even at 37 degrees. A simple functional assay for unwinding protein depends on its essential role in the conversion of phage G4 single-stranded DNA to the replicative form. Unwinding protein stimulates initiation of replication of all single-stranded phage DNAs. Approximately 300 copies of unwinding protein are present per cell, as estimated by antibody titration, an amount sufficient to cover substantial lengths of DNA in several replicating forks.

Bacterial Proteins↗

PubMed1996

Detection of EBER nuclear RNA in T-cell lymphomas involving the skin--an in situ hybridization study.

To examine the relationship of Epstein-Barr virus (EBV) to cutaneous T-cell lymphoma (CTCL), a retrospective study was performed using formalin-fixed paraffin-embedded skin biopsies from 12 patients with cutaneous T-cell lymphoma. All cases were immunophenotyped with a panel of monoclonal antibodies including CD45RO, CD43, MB-2, CD20, and CD68. In situ hybridization studies to detect the EBV genome were performed, using the EBV-RNA probes for the early RNAs of EBV, namely EBER and BHLF. Among the 12 patients, six had primary CTCL, and six had secondary CTCL. The nasal cavity was the most frequent extracutaneous primary site. Immunophenotypically, all of the cases were of the T-cell lineage. In situ hybridization demonstrated a diffuse strong positive reaction for EBV transcripts (EBER) nuclear RNA in five out of 12 cases (41.7%) and a negative reaction for BHLF nuclear RNA probe in all the cases studied. Angiocentric lymphomas were positive in three out of the five cases (60%) studied. Secondary CTCL had a higher correlation with EBV than did primary CTCL, especially primary nasal T-cell lymphoma with subsequent cutaneous involvement. The EBV genome in lymphoma cells existed in a latently infected form but not in an actively replicating form. Three out of the five EBV-positive patients died, 8 months to 4 years after diagnosis. EBV infection alone in the lymphoma does not seem to affect the prognosis of CTCL patients significantly.

Adolescent↗

PubMed1997

The Rep78 gene product of adeno-associated virus (AAV) self-associates to form a hexameric complex in the presence of AAV ori sequences.

The Rep78 and Rep68 proteins of adeno-associated virus (AAV) are replication initiator proteins that bind the viral replicative-form origin of replication, nick the origin in a site- and strand-specific fashion, and mediate vectorial unwinding of the DNA duplex via an ATP-dependent helicase activity, thus initiating a strand displacement mechanism of viral DNA replication. Genetic and biochemical studies have identified Rep mutants that demonstrate a trans-dominant negative phenotype in vitro and in vivo, suggesting the possibility that multimerization of Rep is essential for certain replicative functions. In this study, we have investigated the ability of the largest of the Rep proteins, Rep78, to self-associate in vitro and in vivo. Self-association of Rep78 in vivo was demonstrated through the use of a mammalian two-hybrid system. Rep-Rep protein interaction was confirmed in vitro through coimmunoprecipitation experiments with a bacterially expressed maltose-binding protein-Rep78 fusion protein in combination with [35S]methionine-labeled Rep78 synthesized in a coupled in vitro transcription-translation system. Mapping studies with N- and C-terminal truncation mutant forms of Rep indicate that amino acid sequences required for maximal self-association occur between residues 164 and 484. Site-directed mutagenesis identified two essential motifs within this 321-amino-acid region: (i) a putative alpha-helix bearing a 3,4-hydrophobic heptad repeat reminiscent of those found in coiled-coil domains and (ii) a previously recognized nucleoside triphosphate-binding motif. Deletion of either of these regions from the full-length polypeptide resulted in severe impairment of Rep-Rep interaction. In addition, gel filtration chromatography and protein cross-linking experiments indicated that Rep78 forms a hexameric complex in the presence of AAV ori sequences.

Amino Acid Sequence↗

PubMed1994

Characterization of the temporal accumulation of minute virus of mice replicative intermediates.

We have characterized the temporal appearance and accumulation of minute virus of mice (MVM) replicative forms (RF) in highly synchronized single rounds of infection using a combination of restriction endonuclease analysis and two-dimensional agarose gel electrophoresis. Between 4 and 12 h after release of infected cells into the S-phase, both monomer (mRF) and dimer RF (dRF) increased exponentially at similar rates such that the ratio of mRF relative to dRF remained unchanged. These DNA forms accumulated at a faster rate than MVM RNAs, suggesting that the number of DNA templates available for replication is limiting, not the expression of MVM gene products, and that the majority of DNA templates are likely to be destined for DNA amplification rather than transcription and further gene expression. During this exponential DNA amplification phase, approximately 65% of mRF were in a fully extended form, whereas most of the remaining mRF were covalently closed in the left end and extended in the right end. Although MVM replication presumably generates right-hand turn-around mRF, only a low level of this form persists (5 to 10% of total mRF) at all times examined, suggesting that this form must be quickly converted to the extended form. Greater than 90% of dRF, which have right-hand palindromes on both ends of the molecule, were extended on both ends. A significant proportion of dRF and higher concatemers are nicked in the left-hand palindrome, suggesting that resolution of dRF into two mRFs may occur via single-stranded nicks rather than a double-stranded cut. An additional replicative form, previously termed band X, has been identified as an RNA-DNA duplex. This band is formed predominantly intracellularly, before cell lysis but its biological significance remains unclear. Our results provide direct experimental support for many of the predictions of the current models of parvovirus replication and suggest that the kinetic hairpin transfer model should be adjusted to include a strand-transfer of similar mechanism for the resolution of dRF to account adequately for the production of left-end turn-around forms.

Animals↗

PubMed1985

Active and inactive replication of hepatitis B virus deoxyribonucleic acid in chronic liver disease.

Little is known about the replicative forms of hepatitis B virus (HBV) in the liver in chronic liver disease. We therefore analyzed HBV DNA and the changes in DNA signals after endonuclease digestion in liver tissues taken from 64 patients with hepatitis B surface antigen-positive chronic liver disease. The "active" replication pattern, which included various replicative intermediates, was seen in 36 of 38 (95%) hepatitis B e antigen-seropositive patients. This pattern was also found in 5 of 26 (19%) hepatitis B e antigen-seronegative patients who showed the highest mean serum alanine aminotransferase level (403 +/- 184 mU/ml). Most of them had advanced liver disease. Episomal viral DNA of an "inactive" type having only the supercoiled form was found in 3 patients; they showed the lowest mean serum alanine aminotransferase level (27 +/- 7 mU/ml) and only mild liver disease. As with duck HBV infection, episomal replicative forms of human HBV could be resolved by Southern blot analysis and seem to have clinical implications in human HBV infection.

Adult↗

PubMed1975

Stabilization of promoter complexes with a single ribonucleoside triphosphate.

Under specific binding conditions RNA polymerase forms complexes at several sites of the replicative form DNA of bacteriophage fd. One of these complexes becomes stable to both high salt and low temperature after incubation with GTP. None of the complexes is stabilized by ATP. The stabilization by GTP results from the synthesis of an oligo(G) chain, which is bound in the complex. Size and pyrimidine fingerprints of the DNA segment protected by the enzyme against digestion with DNase are not changed upon initiation of oligo(G) synthesis. This result indicates that binding site and initiation site are identical parts of a promoter region.

Binding Sites↗

PubMed1994

HIV-1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages.

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Q beta, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on production of phage once infection by M13KO7 was established. This result indicated that integrase affects an early stage in infection. Integrase also inhibited phage production following transfection by either single-stranded or double-stranded (replicative form) M13 DNA, it blocked M13 DNA replication, as assayed by incorporation of radioactive nucleotides into DNA, and it failed to affect bacterial pilus function. These data suggest that integrase interacts in vivo with phage nucleic acid, a conclusion supported by studies in which integrase was shown to have a DNA-binding activity in its C-terminal portion. This portion of integrase was both necessary and sufficient for interference of plaque formation by M13 in the present study. Expression of the N-terminal portion of integrase at the same level as intact integrase had little effect on phage growth, indicating that expression of foreign protein in general was not responsible for the inhibitory effect. The simple bacteriophage assay described is potentially useful for identifying integrase mutants that lack single-stranded DNA binding activity.

Antiviral Agents↗

PubMed1972

Replicating, convalently closed, circular DNA from kinetoplasts of Trypanosoma cruzi.

When Trypanosoma cruzi are treated with Berenil, a trypanocide, their kinetoplast DNA contains an increased proportion of double-branched circular molecules. These replicating molecules have closed-circular template strands; their decrease in density when complexed by ethidium bromide in a cesium chloride gradient is proportional to the length of the replicated segments. Replication seems to be blocked at specific points, which are equidistantly spaced along the circular kinetoplast DNA molecules. Analysis of about 800 replicating forms showed that the lengths of the replicated branches are not distributed at random, but into several populations, which correspond to multiples of 15% of the total contour length of 0.5 mum. This distribution evokes a discontinuous replication process. The problem of whether kinetoplast DNA is synthesized by successive replication units, or whether and how Berenil might induce specific blocking of DNA replication, is discussed.

Acetates↗