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At least 1,243 records · Page 69Linked to original sources
PubMed1992

Transient replication of human papillomavirus DNAs.

Information on papillomavirus DNA replication has primarily derived from studies with bovine papillomavirus type 1 (BPV-1). Our knowledge of DNA replication of the human papillomaviruses (HPVs) is quite limited, in part because of the lack of a cell culture system capable of supporting the stable replication of HPV DNA. This study demonstrates that the full-length genomic DNAs of HPV types 11 and 18 (HPV-11 and HPV-18), but not HPV-16, are able to replicate transiently after transfection into several different human squamous cell carcinoma cell lines. This system was used to identify the viral cis and trans elements required for DNA replication. The viral origins of replication were localized to a region of the viral long control region. Like BPV-1, E1 and E2 were the only viral factors required in trans for the replication of plasmids containing the origin. Cotransfection of a plasmid expressing the E1 open reading frame (ORF) from HPV-11 with a plasmid that expresses the E2 ORF from HPV-6, HPV-11, HPV-16, or HPV-18 supported the replication of plasmid DNAs containing the origin regions of HPV-11, HPV-16, or HPV-18, indicating that there are functions shared among the corresponding E1 and E2 proteins and origins of these viruses. Although HPV-16 genomic DNA did not replicate by itself under experimental conditions that supported the replication of HPV-11 and HPV-18 genomic DNAs, expression of the HPV-16 early region functions from a strong heterologous promoter supported the replication of a cotransfected plasmid containing the HPV-16 origin of replication. This finding suggests that the inability of the HPV-16 genomic DNA to replicate transiently in the cell lines tested was most likely due to insufficient expression of the viral E1 and/or E2 genes required for DNA replication.

Base Sequence↗
PubMed1999

Replication, integration, and packaging of plasmid DNA following cotransfection with baculovirus viral DNA.

Infection-dependent replication assays have been used to identify numerous putative origins of baculovirus replication. However, plasmid DNA, when cotransfected into insect cells with Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) DNA, replicates independently of any viral sequence in cis (11). Cotransfection of transfer plasmids and baculovirus DNA is a common procedure used in generating recombinant viruses and in measuring the level of gene expression in transient-expression assays. We have examined the fate of a series of vector plasmids in cotransfection experiments. The data reveal that these plasmids replicate following cotransfection and the replication of plasmid DNA is not due to acquisition of viral putative origin sequences. The conformation of plasmid DNA replicating in the cotransfected cells was analyzed and found to exist as high-molecular-weight concatemers. Ten to 25% of the replicated plasmid DNA was integrated into multiple locations on the viral genome and was present in progeny virions following serial passage. Sequence analysis of plasmid-viral DNA junction sites revealed no homologous or conserved sequences in the proximity of the integration sites, suggesting that nonhomologous recombination was involved during the integration process. These data suggest that while a rolling-circle mechanism could be used for baculovirus DNA replication, recombination may also be involved in this process. Plasmid integration may generate large deletions of the viral genome, suggesting that the process of DNA replication in baculovirus may be prone to generation of defective genomes.

Animals↗
PubMed1982

Initiation and termination of phage f1 plus-strand synthesis.

The origin of DNA replication of bacteriophage f1 contains a nucleotide sequence that is used both for the initiation of viral (plus) strand synthesis and for its termination. With chimeric plasmids containing two f1 functional origins in the same orientation, synthesis of chimeric plus-strand DNA is initiated, after f1 infection, at either one of the two f1 origins and is terminated at the other. Thus, the chimeric plasmids segregate into two replicons, each of them containing only one f1 origin. This system has been used to test several fragments of the f1 origin varying in size or in nucleotide sequence for their ability to function in either initiation or termination of viral strand synthesis. Our data show that the f1 origin is composed of two overlapping but distinct domains (signals), one for initiation and the other for termination of plus-strand synthesis.

Base Sequence↗
PubMed1991

Exonucleolytic proofreading of leading and lagging strand DNA replication errors.

We have asked whether exonucleolytic proofreading occurs during simian virus 40 origin-dependent, bidirectional DNA replication in extracts of human HeLa cells. In addition, we have compared the fidelity of leading and lagging strand DNA synthesis. In a fidelity assay that scores single-base substitution errors that revert a TGA codon in the lacZ alpha gene in an M13mp vector, providing an excess of a single dNTP substrate over the other three dNTP substrates in a replication reaction generates defined, strand-specific errors. Fidelity measurements with two vectors having the origin of replication on opposite sides of the opal codon demonstrate that error rates for two different A.dCTP and T.dGTP mispairs increase when deoxyguanosine monophosphate is added to replication reaction mixtures or when the concentration of deoxynucleoside triphosphates is increased. The data suggest that exonucleolytic proofreading occurs on both strands during bidirectional replication. Measurements using the two simian virus 40 origin-containing vectors suggest that base substitution error rates are similar for replication of the leading and lagging strands.

Antigens, Polyomavirus Transforming↗
PubMed1988

The adeno-associated virus rep gene inhibits replication of an adeno-associated virus/simian virus 40 hybrid genome in cos-7 cells.

A hybrid adeno-associated virus (AAV)/simian virus 40 (SV40) genome is described. In this construct SV40 regulatory sequences, including the early promoter/enhancers and origin of DNA replication, were substituted for the AAV p5 promoter, which normally controls expression of the AAV rep gene. The hybrid genome was phenotypically indistinguishable from wild-type AAV in human cells in the presence or absence of helper virus. Upon transfection into cos-7 cells, which constitutively produced the SV40 tumor antigen, the genome replicated as a plasmid when the SV40 origin was used, although with a low efficiency compared with that of a non-AAV/SV40 replicon. The low level of replication was due to an inhibitory effect of an AAV rep gene product and was specific for replicons containing AAV sequences. Target AAV sequences required for inhibition by rep appeared to reside in the terminal repetitions since deletion of these sequences allowed efficient replication in the presence of the rep gene. The possible role for negative autoregulation of AAV DNA replication in latent infection and helper-dependent replication by AAV is discussed.

Adenoviridae↗
PubMed1988

Bent DNA structures associated with several origins of replication are recognized by a unique enzyme from trypanosomatids.

Sequence-directed bending of the DNA double helix is a conformational variation found in both prokaryotic and eukaryotic organisms. The utilization of bent DNA structures from various sources as specific signals recognized by an enzyme is demonstrated here using a unique endonuclease purified from trypanosomatid cells. Crithidia fasciculata nicking enzyme was previously shown to recognize specifically the bent structure found in kinetoplast DNA minicircles. The binding constant measured for this specific interaction is of two orders of magnitude higher than that measured for the binding of the enzyme to a non-curved sequence. As determined by binding competition and mobility shift electrophoresis analyses, this enzyme recognizes the sequence-directed bends associated with the origins of replication of bacteriophage lambda and simian virus 40 (SV40), as well as that located within the autonomously replicating sequence (ARS1) region of the yeast S. cerevisiae.

Animals↗
PubMed1993

Inhibition of DNA replication factor RPA by p53.

The tumour suppressor p53 specifically interferes with the onset of S phase. The mechanism of the growth suppression action of the protein is unclear, though recent evidence points to transcriptional activation and repression functions of the protein. A competing hypothesis suggests that p53 interacts with the DNA replication apparatus and directly interferes with DNA replication. The major evidence for this hypothesis is that p53 interacts with the simian virus 40 (SV40)-encoded protein T antigen and interferes with the ability of T antigen to unwind the SV40 origin of DNA replication, and recruit DNA polymerase alpha to the replication initiation complex. Here we report that p53 physically interacts with and inhibits the function of a cellular DNA replication factor, the single-stranded DNA-binding protein complex RPA.

Antigens, Polyomavirus Transforming↗
PubMed1996

Complete replication of plasmid DNA containing a single UV-induced lesion in human cell extracts.

To investigate the effect of the major UV-induced lesions on SV40 origin-dependent DNA replication and mutagenesis in a mammalian cell extract, double-stranded plasmids containing a single cis,syn-cyclobutane dimer or a pyrimidine-pyrimidone (6-4) photoproduct at a unique TT sequence have been constructed. These plasmids have been used as templates in DNA replication-competent extracts from human HeLa cells. Plasmids containing a single pyrimidine cyclobutane dimer on the potential lagging strand for DNA replication are replicated with an efficiency approximately equal to that of an unmodified plasmid. A small decrease in replication efficiency of approximately 20% was observed when the lesion was located on the potential leading strand for DNA replication. In both orientations, DpnI-resistant, replicated closed circular plasmid DNA was sensitive to nicking by the pyrimidine dimer-specific enzyme, T4 endonuclease V, indicating that complete replication of the damaged plasmid occurs in vitro. In contrast, a (6-4) photoproduct, within the same site and sequence context on the lagging strand for DNA synthesis, inhibits replication in vitro by an average of approximately 50%, indicating that the mammalian replication complex responds differently to the two major UV-induced lesions during DNA replication in vitro. Analysis of the DpnI-resistant, replicated DNA for mutations targeted to the lesion site indicates that neither of these lesions resulted in significant mutagenesis. UV-induced lesions at TT sites may therefore be poorly mutagenic under these conditions for DNA replication in human cell extracts in vitro.

Base Sequence↗
PubMed1991

Establishment of immortalized cell lines from mouse peritoneal macrophages following transformation with SV40 early region DNA deleted at the origin of replication.

Two murine peritoneal macrophage cell lines have been isolated by transforming primary cells with simian virus 40 (SV40) origin-deleted DNA. These lines have been maintained in continuous culture for over 8 months and have been shown to express macrophage-specific properties throughout this time. The cell lines are F4/80 positive; express Fc receptors; will phagocytose immunoglobulin-coated red cells and latex beads; stain with neutral red; and have non-specific esterase and plasminogen activator activities. Lysozyme, collagenase, prostaglandin E2, acid phosphatase and 5'-nucleotidase activities have also been detected and quantified.

Animals↗
PubMed1995

Cellular factors required for papillomavirus DNA replication.

In vitro replication of papillomavirus DNA has been carried out with a combination of purified proteins and partially purified extracts made from human cells. DNA synthesis requires the viral E1 protein and the papillomavirus origin of replication. The E2 protein stimulates DNA synthesis in a binding site-independent manner. Papillomavirus DNA replication is also dependent on the cellular factors replication protein A, replication factor C, and proliferating-cell nuclear antigen as well as a phosphocellulose column fraction (IIA). Fraction IIA contains DNA polymerase alpha-primase and DNA polymerase delta. Both of these polymerases are essential for papillomavirus DNA replication in vitro. However, unlike the case with T-antigen-dependent replication from the simian virus 40 origin, purified DNA polymerase alpha-primase and delta cannot efficiently replace fraction IIA in the replication reaction. Hence, additional cellular factors seem to be required for papillomavirus DNA replication. Interestingly, replication factor C and proliferating-cell nuclear antigen are more stringently required for DNA synthesis in the papillomavirus system than in the simian virus 40 in vitro system. These distinctions indicate that there must be mechanistic differences between the DNA replication systems of papillomavirus and simian virus 40.

Bovine papillomavirus 1↗
PubMed1984

Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication.

Chimeric SV40 DNA containing only the early region, or plasmid DNA harboring the origin-promoter-enhancer region of SV40, when introduced into CV-1 or Cos-1 monkey cells by DEAE-dextran mediated transfer are rapidly assembled in a typical chromatin structure revealed by the generation of a regular 190 bp repeat ladder after micrococcal nuclease digestion. DNA replication is not required for this assembly process. Chromatin-specific DNase I hypersensitive sites are observed in the enhancer region of these minichromosomes. The pattern of the sites differs between non-replicating and post-replicated chromatin. The latter is identical to that observed in the lytic cycle. The presence of large T antigen is not sufficient for the shift in the structure of the chromatin. These experiments suggest that replication can modulate protein-DNA interactions during viral infection or upon cell differentiation.

Animals↗
PubMed2001

Timely release of both replication forks from oriC requires modulation of origin topology.

Initiation of DNA replication at oriC occurs bidirectionally both in vivo and in vitro. Although the proteins involved in establishing the replication forks are known, little is known about the events that ensure that initiation is bidirectional. We show here that in the absence of DNA gyrase, replication fork progression from oriC on a plasmid template in vitro is unidirectional, although both replication forks have formed at the origin. There was no bias in the release of one fork or the other, ruling out protein blockage of one fork as a possible reason for the asymmetric release. Timely release of both forks required the presence of either DNA gyrase or topoisomerase IV, suggesting that modulation of the topology of the origin region is the governing factor.

Bacterial Proteins↗
PubMed2004

Internalization of exogenous human immunodeficiency virus-1 protein, Tat, by KG-1 oligodendroglioma cells followed by stimulation of DNA replication initiated at the JC virus origin.

JC virus (JCV) is the etiological agent of an opportunistic brain infection, progressive multifocal leukoencephalopathy (PML), in AIDS. PML is fatal in approximately 4% of HIV-infected individuals, and although the overall incidence has fallen due to highly aggressive antiretroviral therapy (HAART), this percent has remained steady. It has been shown that the Tat protein of human immunodeficiency virus-1 (HIV-1) interacts in cells with cellular protein Puralpha. This interaction can stimulate transcription of both HIV-1 and JCV genes. HIV-1, however, infects primarily microglia and astrocytes in the brain, whereas JCV infects primarily oligodendrocytes. Although HIV-1 has been shown capable of infecting oligodendrocytes in vitro (Albright et al., 1996), no instance of viral coinfection of such cells with JCV has been reported. Tat is known to be secreted from cells in which it is made. Here we ask whether such exogenous Tat can influence JCV replication in oligodendrocytes. We find that glial cells infected with either HIV-1 or JCV are in proximity at the outer edge of PML lesions. Exogenous Tat is avidly incorporated into cultured KG-1 oligodendroglioma cells over a 72-h period and is colocalized with endogenous Puralpha both nuclear and juxtanuclear. At concentrations in the medium well below the pM range, Tat stimulates several-fold the replication in vivo of DNA initiated at the JCV origin. These results define a pathway by which a protein made by HIV-1 can directly affect the course of infection by another disease-causing virus.

Cell Line, Tumor↗
PubMed1988

Mouse p53 blocks SV40 DNA replication in vitro and downregulates T antigen DNA helicase activity.

Immunopurified mouse p53 proteins were used to gain experimental access to the mechanisms underlying nonprimate p53 directed suppression of SV40 origin directed DNA replication in vivo. In replication competent HeLa cell extracts containing exogenous T antigen, mouse p53 blocks T antigen dependent DNA synthesis as in vivo. However, in transcription competent HeLa extracts, mouse p53 has no effect either on overall transcription or on the ability of immunopurified T antigen to downregulate SV40 early transcription. We show that although mouse p53 has no significant effect on T antigen encoded activities such as ATPase and DNA binding, helicase activity is somewhat reduced suggesting that the in vivo suppression by mouse p53 of SV40 replication may be due, at least in part, to direct modulation of T antigen function.

Adenosine Triphosphatases↗
PubMed1994

Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro.

Complete enzymatic replication of DNA from the simian virus 40 origin has been reconstituted with T antigen and highly purified cellular proteins. DNA polymerase-alpha/primase functions primarily to synthesize RNA-DNA primers for initiation of DNA replication at the origin and for priming each Okazaki fragment. A polymerase switching mechanism requiring replication factor C and the proliferating cell nuclear antigen allows two molecules of DNA polymerase-delta to replicate both strands of the double helix conjointly.

Animals↗
PubMed1988

Cellular factors required for multiple stages of SV40 DNA replication in vitro.

Plasmids containing the SV40 origin replicate in the presence of SV40 T antigen and a cell free extract derived from human 293 cells. Upon fractionation of this extract, two essential replication factors have been identified. One of these is a multi-subunit DNA binding protein containing polypeptides of 70,000, 34,000 and 11,000 daltons which may function as a eukaryotic single strand DNA binding protein (SSB). The other partially purified fraction is required with T antigen for the first stage of DNA replication, the formation of a pre-synthesis complex at the replication origin. These results, and others, define multiple stages of SV40 DNA replication in vitro which are analogous to multiple stages of Escherichia coli and phage lambda replication, and may reflect similar events in the replication of cellular chromosomes.

Antigens, Polyomavirus Transforming↗
PubMed1998

DNA binding of polyomavirus large T-antigen: kinetics of interactions with different types of binding sites.

Polyomavirus large T-antigen binds to GRGGC sites in double-stranded viral DNA, regulating transcription and replication. Using surface plasmon resonance to record interactions of large T-antigen with different types of binding sites, we found that the configuration of recognition motifs influenced both the association and dissociation rates. Particularly, the complex formed at the origin of DNA replication was labile. A comparison of the interactions between large T-antigen and binding sites with one, two and four GRGGC motifs in tandem showed a strong preference for dimer binding, without detectable co-operativity between dimers. Sodium chloride stabilised the complexes, whereas the dissociation increased rapidly by increasing pH above 7.0.

Antigens, Viral, Tumor↗