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PubMed1990

DNA-replication recovery inhibition and subsequent reinitiation in UV-radiation-damaged E. coli: a strategy for survival.

Using the incorporation of [14C]thymine to measure DNA accumulation, it was shown that exposure of the B/r strain of Escherichia coli to 10 J/m2 of ultraviolet radiation (UV) inhibits replication for about 20 min, but then resumption of replication occurs. Pulse-labelling with [3H]thymidine after exposure of the WT strain to this fluence confirmed the transient inhibition and recovery of DNA replication. After recovery, the rate of accumulation of DNA in the culture increases, to exceed that of the exponentially growing culture, so that eventually the amount of DNA almost equals that of the unirradiated culture. After a higher fluence (20 J/m2), an inhibition of replication recovery was revealed. This fluence delays the reinitiation of DNA accumulation in the culture, measured by [14C]thymine incorporation, for 25 min more, in addition to the 20-min recovery period. This finding was confirmed with pulse-labelling studies, which revealed that the higher exposure represses the rates of replication for 45 min before replication at the normal rate reinitiates in the culture. It was proposed that the inhibition of recovery revealed by these investigations is effected by the UV-induction of an active DNA-replication recovery-inhibition process. With the uvrA strain, rate studies revealed that 1.5 J/m2 of UV (a reduced fluence necessary because of the greater sensitivity of the strain) induces a transient inhibition of DNA replication, with considerable recovery following. Exposure to 3.0 J/m2 induces the transient inhibition of replication, followed by massive recovery inhibition after 20 min of incubation. With uvrA recA, both the lower and the higher fluence resulted in an immediate block of replication with no recovery, confirming the recA gene dependency of the recovery process. The decrease in rate of replication comparable to that seen in the uvrA strain after 20 min, and taken as evidence of the function of the recovery-inhibition process, was not seen. The evidence supports the concept that a process somehow triggered by higher UV fluences functions to repress replication temporarily, presumably allowing time for repair processes to take place before replication overruns closely linked pyrimidine dimers on opposite strands to create lethal lesions.

DNA Replication↗
PubMed2002

Allele-specific replication associated with aneuploidy in blood cells of patients with hematologic malignancies.

We hypothesize that coordination between the two DNA parental sets in somatic cells is essential for the stability of the diploid genome, and that its disruption is associated with the many alterations observed in the various cancerous phenotypes. As coordination between two allelic counterparts is well exemplified by synchrony in replication timing, we examined, in blood cells of patients suffering from various hematologic malignancies, replication patterns of five loci. These loci were three cancer-implicated genes (TP53, AML1, and RB1) and two nontranscribed sequences engaged in chromosome segregation. All five loci normally display synchrony in allelic replication timing. In addition, in order to exemplify an asynchronous mode of allelic replication, we followed the replication of allelic counterparts of an imprinted gene (SNRPN), which is distinguished by its asynchronous mode of allelic replication (allele-specific replication). Allelic replication patterns were studied by fluorescence in situ hybridization (FISH), which has been shown to distinguish between nonreplicated and replicated regions of the genome in interphase cells, based on the structure of the specific hybridization signals that are being detected. Using the FISH replication assay we observed, for all loci which normally exhibit synchrony in allelic replication, loss of synchrony when present in blood cells of patients with hematologic malignancies. The loss of synchrony in allelic replication in patients' cells was accompanied by aneuploidy (chromosome losses and gains), the hallmark of cancer. We were able to reinstate the normal pattern of replication in the patients' cells by introducing an inhibitor of DNA methylation. It thus appears loss of allelic coordination is an epigenetic alteration characterizing cancer, which is easily identified by simple cytogenetic means and has a potential use in both cancer investigation and detection.

Adolescent↗
PubMed1994

E1 protein of human papillomavirus type 1a is sufficient for initiation of viral DNA replication.

Previous studies on transient replication of papillomaviruses have shown an absolute requirement for the viral E1 and E2 proteins in DNA replication. Here we demonstrate that for human papillomavirus type 1a (HPV-1a) DNA, the E1 protein alone is sufficient for in vivo replication of plasmids containing the viral origin of replication. Replication was origin-specific and required the presence of a DNA sequence containing a putative E1 binding site, but the E2 binding sites were dispensable. In the presence of the E1 protein, E2 stimulated replication of plasmids containing the E1 and E2 binding sites, but no stimulation was observed when the origin plasmids lacked E2 binding sites. Conversely, in the presence of E1 alone, the E2 binding sites did not affect replication. Plasmids containing the replication origins of HPV-6b, HPV-18, and bovine papillomavirus type 1 (BPV-1) also replicated efficiently in the presence of the HPV-1a E1 and E2 proteins. However, plasmids containing the origins of HPV-6b and HPV-18 failed to replicate in the presence of HPV-1a E1 alone, whereas a plasmid containing the BPV-1 origin replicated to lower levels than the HPV-1a origin-containing plasmid. These results suggest that replication from papillomaviral origins in the presence of E1 alone is presumably dependent on the strength of E1-origin interactions. Additionally, E1-dependent replication is stimulated by the E2 protein in the presence of E2 binding sites.

Base Sequence↗
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↗
PubMed1998

Mechanistic studies on the impact of transcription on sequence-specific termination of DNA replication and vice versa.

Since DNA replication and transcription often temporally and spatially overlap each other, the impact of one process on the other is of considerable interest. We have reported previously that transcription is impeded at the replication termini of Escherichia coli and Bacillus subtilis in a polar mode and that, when transcription is allowed to invade a replication terminus from the permissive direction, arrest of replication fork at the terminus is abrogated. In the present report, we have addressed four significant questions pertaining to the mechanism of transcription impedance by the replication terminator proteins. Is transcription arrested at the replication terminus or does RNA polymerase dissociate from the DNA causing authentic transcription termination? How does transcription cause abrogation of replication fork arrest at the terminus? Are the points of arrest of the replication fork and transcription the same or are these different? Are eukaryotic RNA polymerases also arrested at prokaryotic replication termini? Our results show that replication terminator proteins of E. coli and B. subtilis arrest but do not terminate transcription. Passage of an RNA transcript through the replication terminus causes the dissociation of the terminator protein from the terminus DNA, thus causing abrogation of replication fork arrest. DNA and RNA chain elongation are arrested at different locations on the terminator sites. Finally, although bacterial replication terminator proteins blocked yeast RNA polymerases in a polar fashion, a yeast transcription terminator protein (Reb1p) was unable to block T7 RNA polymerase and E. coli DnaB helicase.

Bacillus subtilis↗
PubMed1999

Involvement of DNA-dependent protein kinase in UV-induced replication arrest.

Cells exposed to UV irradiation are predominantly arrested at S-phase as well as at the G(1)/S boundary while repair occurs. It is not known how UV irradiation induces S-phase arrest and yet permits DNA repair; however, UV-induced inhibition of replication is efficiently reversed by the addition of replication protein A (RPA), suggesting a role for RPA in this regulatory event. Here, we show evidence that DNA-dependent protein kinase (DNA-PK), plays a role in UV-induced replication arrest. DNA synthesis of M059K (DNA-PK catalytic subunit-positive (DNA-PKcs(+))), as measured by [(3)H]thymidine incorporation, was significantly arrested by 4 h following UV irradiation, whereas M059J (DNA-PKcs(-)) cells were much less affected. Similar results were obtained with the in vitro replication reactions where immediate replication arrest occurred in DNA-PKcs(+) cells following UV irradiation, and only a gradual decrease in replication activity was observed in DNA-PKcs(-) cells. Reversal of replication arrest was observed at 8 h following UV irradiation in DNA-PKcs(+) cells but not in DNA-PKcs(-) cells. Reversal of UV-induced replication arrest was also observed in vitro by the addition of a DNA-PK inhibitor, wortmannin, or by immunodepletion of DNA-PKcs, supporting a positive role for DNA-PK in damage-induced replication arrest. The RPA-containing fraction from UV-irradiated DNA-PKcs(+) cells poorly supported DNA replication, whereas the replication activity of the RPA-containing fraction from DNA-PKcs(-) cells was not affected by UV, suggesting that DNA-PKcs may be involved in UV-induced replication arrest through modulation of RPA activity. Together, our results strongly suggest a role for DNA-PK in S-phase (replication) arrest in response to UV irradiation.

Androstadienes↗
PubMed1988

Do transcriptional enhancers also augment DNA replication?

Enhancers are DNA elements that augment transcription in cis, independent of distance and orientation. Evidence such as hormone dependent neoplastic cell growth and the stimulation of viral replication by sequences present in enhancers suggests that enhancers may also directly affect DNA replication. We tested this hypothesis in recombinant plasmids by asking whether sequences that stimulated DNA replication shared the properties of transcriptional enhancers. The homologous simian virus 40 (SV40) core enhancer was ligated either adjacent to or 2.6 kb distant from the SV40 minimal origin of replication (ori) in both orientations. Plasmids were transfected into T antigen producing COS cells, and episomal DNA was harvested for quantitation of replication. Replication could be assessed either as accumulation of fmol of MboI sensitive progeny DNA, or as a transition in % DNA in replicated (MboI sensitive) versus unreplicated (DpnI sensitive) form. The two measures were related exponentially (r = 0.86). The SV40 enhancer augmented replication 1.5-10 fold. The effect was time dependent, distance dependent (only the adjacent enhancer locus stimulated replication), partially orientation dependent, and enhancer copy number independent. Phorbol ester did not affect replication. The heterologous glucocorticoid enhancer had no effect on replication. We conclude that the SV40 enhancer's cis-effect on replication seems to be dependent on the close proximity to the replication origin of specific homologous sequences within the enhancer, rather than a typical enhancer-like effect.

Animals↗
PubMed1987

Tissue-specific replication of Friend and Moloney murine leukemia viruses in infected mice.

We have studied the replication of ecotropic murine leukemia viruses (MuLV) in the spleens and thymuses of mice infected with the lymphocytic leukemia-inducing virus Moloney MuLV (M-MuLV), with the erythroleukemia-inducing virus Friend MuLV (F-MuLV), or with in vitro-constructed recombinants between these viruses in which the long terminal repeat (LTR) sequences have been exchanged. At 1 week after infection both the parents and the LTR recombinants replicated predominantly in the spleens with only low levels of replication in the thymus. At 2 weeks after infection, the patterns of replication in the spleens and thymuses were strongly influenced by the type of LTR. Viruses containing the M-MuLV LTR exhibited a remarkable elevation in thymus titers which frequently exceeded the spleen titers, whereas viruses containing the F-MuLV LTR replicated predominantly in the spleen. In older preleukemic mice (5 to 8 weeks of age) the structural genes of M-MuLV or F-MuLV predominantly influenced the patterns of replication. Viruses containing the structural genes of M-MuLV replicated efficiently in both the spleen and thymus, whereas viruses containing the structural genes of F-MuLV replicated predominantly in the spleen. In leukemic mice infected with the recombinant containing F-MuLV structural genes and the M-MuLV LTR, high levels of virus replication were observed in splenic tumors but not in thymic tumors. This phenotypic difference suggested that tumors of the spleen and thymus may have originated by the independent transformation of different cell types. Quantification of polytropic MulVs in late-preleukemic mice infected with each of the ecotropic MuLVs indicated that the level of polytropic MuLV replication closely paralleled the level of replication of the ecotropic MuLVs in all instances. These studies indicated that determinants of tissue tropism are contained in both the LTR and structural gene sequences of F-MuLV and M-MuLV and that high levels of ecotropic or polytropic MuLV replication, per se, are not sufficient for leukemia induction. Our results further suggested that leukemia induction requires a high level of virus replication in the target organ only transiently during an early preleukemic stage of disease.

Animals↗
PubMed1992

Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells.

Full-length and subgenomic poliovirus RNAs were transcribed in vitro and transfected into HeLa cells to study viral RNA replication in vivo. RNAs with deletion mutations were analyzed for the ability to replicate in either the absence or the presence of helper RNA by using a cotransfection procedure and Northern (RNA) blot analysis. An advantage of this approach was that viral RNA replication and genetic complementation could be characterized without first isolating conditional-lethal mutants. A subgenomic RNA with a large in-frame deletion in the capsid coding region (P1) replicated more efficiently than full-length viral RNA transcripts. In cotransfection experiments, both the full-length and subgenomic RNAs replicated at slightly reduced levels and appeared to interfere with each other's replication. In contrast, a subgenomic RNA with a similarly sized out-of-frame deletion in P1 did not replicate in transfected cells, either alone or in the presence of helper RNA. Similar results were observed with an RNA transcript containing a large in-frame deletion spanning the P1, P2, and P3 coding regions. A mutant RNA with an in-frame deletion in the P1-2A coding sequence was self-replicating but at a significantly reduced level. The replication of this RNA was fully complemented after cotransfection with a helper RNA that provided 2A in trans. A P1-2A-2B in-frame deletion, however, totally blocked RNA replication and was not complemented. Control experiments showed that all of the expected viral proteins were both synthesized and processed when the RNA transcripts were translated in vitro. Thus, our results indicated that 2A was a trans-acting protein and that 2B and perhaps other viral proteins were cis acting during poliovirus RNA replication in vivo. Our data support a model for poliovirus RNA replication which directly links the translation of a molecule of plus-strand RNA with the formation of a replication complex for minus-strand RNA synthesis.

Cell-Free System↗
PubMed1995

Requirements for the self-directed replication of flock house virus RNA 1.

The larger segment (RNA 1) of the bipartite, positive-sense RNA genome of the nodavirus flock house virus encodes the viral RNA-dependent RNA polymerase. Two nonstructural viral proteins are made during the self-directed replication of this RNA: protein A (110 kDa), the translation product of RNA 1 itself, and protein B (11 kDa), the translation product of a subgenomic RNA (RNA 3) that is produced from RNA 1 during replication. To examine the roles of these proteins in RNA replication, specialized T7 transcription plasmids that contained wild-type or mutant copies of flock house virus RNA 1 cDNA were constructed and used in cells infected with the vaccinia virus-T7 RNA polymerase recombinant to make full-length transcripts that directed their own replication. Sequences in the primary transcripts that extended beyond the ends of the authentic RNA 1 sequence inhibited self-directed RNA replication, but plasmids that were constructed to minimize these terminal extensions produced primary transcripts that replicated as abundantly as authentic RNA 1. Truncation or mutation of the open reading frame for protein A eliminated self-directed replication, although the mutant RNA 1 remained a competent template for replication by wild-type protein A supplied in trans. These results showed that protein A was essential for RNA replication and that the process was not inseparably coupled to complete translation of the template. In contrast, protein B could be eliminated without inhibiting replication by mutations that disrupted the second of the two overlapping open reading frames on RNA 3. Furthermore, a mutant of RNA 1 in which the first nucleotide of the RNA 3 region was changed from G to U replicated at levels as high as those of the wild type without making either RNA 3 or protein B. However, diminishing replication levels were observed during subsequent replicative passages of RNA from both the mutants that could not make protein B. Roles for this protein that could account for the subtle phenotype of these mutants are discussed.

Animals↗
PubMed1999

Regulation of RNA synthesis by the genomic termini of vesicular stomatitis virus: identification of distinct sequences essential for transcription but not replication.

The RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV), a nonsegmented negative-strand RNA virus, directs two discrete RNA synthetic processes, transcription and replication. Available evidence suggests that the two short extragenic regions at the genomic termini, the 3' leader (Le) and the complement of the 5' trailer (TrC), contain essential signals for these processes. We examined the roles in transcription and replication of sequences in Le and TrC by monitoring the effects of alterations to the termini of subgenomic replicons, or infectious viruses, on these RNA synthetic processes. Distinct elements in Le were found to be required for transcription that were not required for replication. The promoter for mRNA transcription was shown to include specific sequence elements within Le at positions 19 to 29 and 34 to 46, a separate element at nucleotides 47 to 50, the nontranscribed leader-N gene junction. The sequence requirements for transcription within the Le region could not be supplied by sequences found at the equivalent positions in TrC. In contrast, sequences from either Le or TrC functioned well to signal replication, indicating that within the confines of the VSV termini, the sequence requirements for replication were less stringent. Deletions engineered at the termini showed that the terminal 15 nucleotides of either Le or TrC allowed a minimal level of replication. Within these confines, levels of replication were affected by both the extent of complementarity between the genomic termini and the involvement of the template in transcription. In agreement with our previous observations, increasing the extent of complementarity between the natural termini increased levels of replication, and this effect was most operative at the extreme genome ends. In addition, abolishing the use of Le as a promoter for transcription enhanced replication. These analyses (i) identified signals at the termini required for transcription and replication and (ii) showed that Le functions as a less efficient promoter for replication than TrC at least in part because of its essential role in transcription. Consequently, these observations help explain the asymmetry of VSV replication which results in the synthesis of more negative- than positive-sense replication products in infected cells.

Base Sequence↗
PubMed2002

Induction of the bovine papillomavirus origin "onion skin"-type DNA replication at high E1 protein concentrations in vivo.

We have studied the replication of plasmids composed of bovine papillomavirus type 1 (BPV1) origin of replication and expression cartridges for viral proteins E1 and E2 in hamster and mouse cells. We found that the replication mode changed dramatically at different expression levels of the E1 protein. At high levels of the E1 protein, overreplication of the origin region of the plasmid was observed. Analysis of the replication products by one-dimensional and two-dimensional gel electrophoresis suggested that initially "onion skin"-type replication intermediates were generated, presumably resulting from initiation of the new replication forks before the leading fork completed the synthesis of the DNA on the episomal plasmid. These replication intermediates served as templates for generation of a heterogeneous set of origin region-containing linear fragments by displacement synthesis at the partially replicated plasmid. Additionally, the linear fragments may have been generated by DNA break-up of the onion skin-type intermediates. Analysis of replication products indicated that generated linear fragments recombined and formed concatemers or circular molecules, which presumably were able to replicate in an E1- and E2-dependent fashion. At moderate and low levels of E1, generated by transcription of the E1 open reading frame using weaker promoters, DNA replication was initiated at much lower levels, which allowed elongation of the replication fork starting from the origin to be more balanced and resulted in the generation of full-sized replication products.

Animals↗
PubMed1989

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.

Animals↗
PubMed2026

Host-interferon-stimulated gene response to virus-host recombinant variants of hepatitis E virus and enhanced viral replication.

The hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. HEV infection can become chronic in immunocompromised individuals, in whom virus-host recombinant variants (VHRVs) can be detected. These variants often harbor host-derived insertions in the polyproline-rich region (PPR), and most display enhanced replication in vitro. However, the mechanisms underlying this replicative advantage remain unclear. It is likely that genes of the infected cells are differentially expressed according to the replicative capacity of the strain. The host factors involved in the improvement of the replicative capacity of these VHRVs are yet to be identified.In this study, we analyzed the host transcriptional response to seven VHRVs in HepG2/C3A cells using bulk RNA sequencing at 48 h and 168 h post-infection. Five VHRVs (RNF19A, ZNF787, KIF1B, RPS17, EEF1A1) previously associated with a high replication rate induced more significant, distinct transcriptomic changes than low-replicative variants (RNA18, RPL6), particularly at 168 h. A shared set of 25 genes, especially interferon-stimulated genes (ISGs), was upregulated in cells infected with high-replicating variants. Interestingly, ISG induction was limited at 48 h despite high viral RNA concentrations, suggesting a delayed antiviral response. At 168 h, high ISG expression coincided with high viral loads, indicating that VHRVs may evade or exploit immune defenses. Our findings reveal candidate ISGs such as IFIT1 and ISG15 that may influence HEV persistence and immune escape. These results offer new insights into the interplay between VHRV replication and host immunity.IMPORTANCEHepatitis E virus (HEV) is a major cause of acute hepatitis and can cause chronic infections in immunocompromised individuals. Virus-host recombinant variants (VHRVs) having integrated host-derived insertions often replicate more effectively, yet the host determinants of this phenotype remain unclear. With RNA sequencing of HepG2/C3A-infected cells, we observed that high-replicating VHRVs induce a delayed but strong expression of interferon-stimulated genes (ISGs), including IFIT1 and ISG15, despite high viral loads. These results suggest that VHRVs may transiently modulate or evade aspects of host antiviral defenses. Our study revealed host transcriptional patterns associated with enhanced viral replication, providing insight into potential mechanisms that enhance HEV replication and highlighting candidate pathways that could influence the interplay between viral replication and immune responses, all requiring further investigation.

Humans↗
PubMed2001

Comparative analyses of transgene delivery and expression in tumors inoculated with a replication-conditional or -defective viral vector.

Viral vectors for cancer can be classified into those that do not replicate (replication-defective vectors) and those that selectively replicate in neoplastic cells (replication-conditional or oncolytic vectors). Both of these can deliver anticancer cDNAs for therapeutic purposes. Opposite hypotheses can be made regarding the advantages of each vector type with regard to anatomic transgene expression. For the former vector, because cDNA delivery occurs in neoplastic cells that have the ability to migrate into the tumor mass, relatively extensive anatomic and temporal expression of anticancer functions may occur. For the latter vector, active viral replication may permit anatomically and temporally extensive delivery of the foreign cDNA into the tumor mass. Herein, we performed a simple comparative analysis to test which of these hypotheses is valid. Direct inoculation of s.c. tumors with a replication-conditional or a replication-defective viral vector, each of which expressed lacZ cDNA, was performed. Tumors were excised and analyzed for anatomic delivery of beta-galactosidase and for neoplastic viral titers. We find that lacZ cDNA expression is observed in approximately 40% of the tumor area 3, 7, and 14 days after injection with the replication-conditional vector, whereas approximately 10% of the tumor area expresses the transgene 3 days after injection with the replication-defective vector, with a rapid decline in expression thereafter. Titers of the replication-conditional virus remain stable within injected tumors for the 14 days of the assay (approximately 1:1,000 of the initial injection dose), whereas titers of the replication-defective vector decrease rapidly after injection (to a value of 1:100,000 of the initial injection dose). Taken in conjunction, these studies show that transgene delivery and expression in tumors last longer and are found throughout an anatomically more extensive area after injection with replication-conditional gene therapy vectors than after injection with replication-defective gene therapy vectors.

Animals↗
PubMed1987

In vitro initiation of DNA replication in simian virus 40 chromosomes.

A soluble system has been developed that can initiate DNA replication de novo in simian virus 40 (SV40) chromatin isolated from virus-infected monkey cells as well as in circular plasmid DNA containing a functional SV40 origin of replication (ori). Initiation of DNA replication in SV40 chromatin required the soluble fraction from a high-salt nuclear extract of SV40-infected cells, a low-salt cytosol fraction, polyethylene glycol, and a buffered salts solution containing all four standard deoxyribonucleoside triphosphates. Purified SV40 large tumor antigen (T-ag) partially substituted for the high-salt nucleosol, and monoclonal antibodies directed against SV40 T-ag inhibited DNA replication. Replication began at ori and proceeded bidirectionally to generate replicating DNA intermediates in which the parental strands remained covalently closed, as observed in vivo. Partial inhibition of DNA synthesis by aphidicolin resulted in accumulation of newly initiated replicating intermediates in this system, a phenomenon not observed under conditions that supported completion of replication only. However, conditions that were optimal for initiation of replication repressed conversion of late-replicating intermediates into circular DNA monomers. Most surprising was the observation that p-n-butylphenyl-dGTP, a potent and specific inhibitor of DNA polymerase-alpha, failed to inhibit replication of SV40 chromatin under conditions that completely inhibited replication of plasmid DNA containing the SV40 ori and either purified or endogenous DNA polymerase-alpha activity. In contrast, all of these DNA synthesis activities were inhibited equally by aphidicolin. Therefore, DNA replication in mammalian cells is carried out either by DNA polymerase-alpha that bears a unique association with chromatin or by a different enzyme such as DNA polymerase-delta.

Antigens, Neoplasm↗
PubMed1984

Evidence for participation of a multiprotein complex in yeast DNA replication in vitro.

Fractions containing a high molecular weight form (Mr approximately equal to 2 X 10(6] of the activity that replicates in vitro both the 2-micron yeast DNA plasmid and the chromosomal autonomously replicating sequence ars 1 can be prepared from cells of the budding yeast Saccharomyces. Protein complexes from the fractions associate in vitro with the replication origins of these DNA elements, as determined by electron microscopy. In the present study, the high molecular weight replicative fraction has been characterized in further detail. The DNA synthetic activity in the high molecular weight fraction was bound to the DNA and could be isolated with it. This binding of the replicating activity to the DNA was greatly reduced in the absence of the 2-micron origins of replication. Association of the protein complexes with DNA depended on the amount of replicating activity added, was sensitive to 0.2 M KCl, and exhibited a requirement for rATP and deoxyribonucleoside triphosphates. It was not blocked, however, by the DNA polymerase inhibitor aphidicolin or by the RNA polymerase inhibitor alpha-amanitin. The lack of inhibition by aphidicolin suggests that the deoxyribonucleoside triphosphates may function as cofactors in the binding of protein complexes to DNA or as substrates for a polymerizing activity such as a primase. Binding of the protein complexes as well as actual DNA replication were heat sensitive in the high molecular weight fraction prepared from the temperature-sensitive mutant of the cell division cycle cdc 8. This suggests that the cdc 8 gene product is present in a replicative protein complex and strengthens the conclusion that the presence of the protein complexes on the DNA is associated with replication. Using independent enzyme assays, several other possible replication proteins (including DNA polymerase I, DNA ligase, DNA primase, and DNA topoisomerase II) have been identified directly in the high molecular weight replicative fraction. All of these results provide support for the idea that a protein complex (or replisome ) is involved in the replication of both the extrachromosomal 2-micron DNA and chromosomal DNA in yeast.

DNA Replication↗
PubMed1997

A cell-free replication system for human polyomavirus JC DNA.

The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and is the etiologic agent of progressive multifocal leukoencephalopathy. In this report, we describe the establishment of a soluble cell-free system that is capable of replicating exogenous plasmid DNA containing the JCV origin of replication. Replication in this system is completely dependent on the addition of JCV large T antigen (TAg). To prepare JCV TAg for replication analysis, a recombinant baculovirus containing the JCV TAg-coding sequence was generated. TAg expressed in insect cells was purified by metal chelate chromatography. JCV TAg supported initiation of JCV DNA replication in the presence of DNA polymerase alpha-primase, replication protein A, and topoisomerase I in a dose-dependent manner and was also capable of supporting DNA replication in crude human cell extracts. Point mutation of TAg-binding site I strongly diminished TAg binding and concomitantly reduced JCV DNA replication in vivo and in vitro by approximately 50%. Point mutation of TAg-binding site II or deletion of the early palindrome completely abolished replication of JCV origin-containing plasmid DNA in vivo and in vitro, marking these sequences as essential components of the JCV core origin. A comparison of several TAgs showed that simian virus 40 TAg, but not mouse polyomavirus (PyV) TAg, supported replication of a plasmid containing a JCV origin. These findings provide evidence that replication in the cell-free system faithfully mimics JCV DNA replication in vivo. Therefore, it may be a useful tool for future analysis of interactions between JCV and its host cell.

Animals↗